CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No.

62/909,585, filed October 2, 2019, which is incorporated by reference herein in its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submitted via EFS-Web to the United States Patent and Trademark Office as an ASCII text file entitled “Seq_List-0235- 000287_ST25.txt” having a size of 7 kilobytes and created on September 23, 2020. The information contained in the Sequence Listing is incorporated by reference herein.

GOVERNMENT FUNDING

This invention was made with government support under R01AI123383 and P41GM103490 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

HIV has been a major threat to human health and a protective AIDS vaccine does not exist. Isolation and characterization of protective antibodies from virus infected individuals targeting the viral envelope glycoprotein was a milestone in fighting against this deadly virus. Since then, inducing the production of protective antibodies through vaccination has been a highly promising strategy to halt the spread of the disease. However, the design of the current generation of vaccines does not maximize stimulation of helper T cells and induce T cell- dependent high-affinity, long-lasting and protective antibody responses against the viral envelope. SUMMARY OF THE APPLICATION

The inventors have identified and characterized a new mechanism for CD4+ T cell- mediated humoral immune responses induced by HIV-envelope glycoprotein. HIV envelope glycoprotein (gp) 120 is heavily glycosylated with 23 to 26 N-glycosylation sites. A CD4+ T cell repertoire recognizes glycopeptide epitopes of gpl20 presented by major histocompatibility class II (MHCII) complex. The processing of gpl20 in the endolysosomes of antigen presenting cells (APCs) yielded glycopeptide epitopes that bind MHCII proteins, stimulate T cells, and induce the production of antibody.

Provided herein are isolated glycopeptides. In one embodiment, a glycopeptide includes an amino acid sequence having structural similarity to an amino acid sequence of at least 7 consecutive amino acids of a human HIV-gpl20 protein. An example of a glycopeptide is one that includes an amino acid sequence having structural similarity to the amino acid sequence LDVVPIDNNNTSY (SEQ ID NO: 1), wherein the residue at position 10 of SEQ ID NO: 1 includes a glycan. In one embodiment, a glycopeptide having structural similarity to SEQ ID NO: 1 further includes a Lysine at the amino-terminal end of SEQ ID NO: 1, an arginine at the carboxy-terminal end of SEQ ID NO:l, or both lysine and arginine. In one embodiment, a glycopeptide includes (i) an amino acid sequence of at least 7 to no greater than 20 consecutive amino acids of a HIV-1 gpl20 protein or (ii) an amino acid sequence having structural similarity to at least 7 to no greater than 20 consecutive amino acids of a HIV-1 gpl20 protein, wherein the glycopeptide includes at least one glycan and glycosylation site, and wherein the glycopeptide binds a MHCII molecule. Other examples of glycopeptides are shown in Table 1.

In one embodiment, the glycopeptide includes no greater than 20 amino acids. In one embodiment, the glycopeptide includes a mannose, such as 2, 3, 4 or 5 mannose residues. In one embodiment, the glycopeptide reacts with mannose-specific lectin Concanavalin A. In one embodiment, the glycan does not include one or more of a fucose saccharide or a sialic acid. In one embodiment, the glycan is an N-linked glycosylation. In one embodiment, the glycopeptide is a multimer. In one embodiment, the glycopeptide is a fusion protein including a heterologous amino acid sequence, such as, but not limited to, a linker sequence. In one embodiment, the heterologous amino acid sequence includes a cleavable sequence, such as, but not limited to, an acid-labile sequence or a protease recognition sequence.

Also provided is a composition that includes a glycopeptide described herein. In one embodiment, the composition includes a pharmaceutically acceptable carrier. In one embodiment, the composition includes an adjuvant.

Further provided is a genetically engineered cell that includes a glycopeptide described herein, for instance, the cell can include a polynucleotide that encodes the peptide that is then processed by the cell to result in the glycopeptide. In one embodiment, the cell includes a N- acetylglucosaminyltransferase I mutation (GnTI-/-). In one embodiment, the stably expresses the glycopeptide.

Also provided are methods. In one embodiment, a method includes administering to a subject an amount of composition that includes a glycopeptide described herein in an amount effective to induce in the subject an immune response to the glycopeptide. In one embodiment, the immune response includes the production of antibody that specifically binds to the glycopeptide. In one embodiment, the immune response includes the production of T cells that produce interleukin-4 and/or interferon-gamma after stimulation by the glycopeptide. In one embodiment, the subject has or is at risk of having an infection caused by HIV.

A method can include treating an infection in a subject. In one embodiment, the method includes administering an effective amount of a composition described herein to a subject having or at risk of having an infection caused by HIV, such as HIV-1.

A method can include treating a symptom in a subject. In one embodiment, the method includes administering an effective amount of the composition described herein to a subject having or at risk of having an infection caused by HIV, such as HIV-1.

A method can include treating a condition in a subject. In one embodiment, the method includes administering an effective amount of composition described herein to a subject having or at risk of having a condition caused by HIV, such as HIV-1.

Terms used herein will be understood to take on their ordinary meaning in the relevant art unless specified otherwise.

The term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements. The words “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

The terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

It is understood that wherever embodiments are described herein with the language “include,” “includes,” or “including,” and the like, otherwise analogous embodiments described in terms of “consisting of’ and/or “consisting essentially of’ are also provided. The term “consisting of’ means including, and limited to, whatever follows the phrase “consisting of.”

That is, “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. The term “consisting essentially of’ indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.

Conditions that are “suitable” for an event to occur, or “suitable” conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event.

Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE FIGURES

The following detailed description of illustrative embodiments of the present disclosure may be best understood when read in conjunction with the following figures.

FIG. 1A-B shows Generation of a gpl20 glycopeptide pool and immunization scheme. (FIG. 1 A) A pool of individual gly copeptides was produced from full-length gpl20 with the use of protease Glu-C. Digested products were separated by chromatography. Glycopeptidecontaining fractions were identified by lectin blot and were pooled together to immunize mice. Production of gpl20-specific IgG was detected by ELISA. (FIG. IB) gpl20 was treated with PNGase F to remove N-glycans or was not treated. The molecular-weight shift of the proteins before and after glycosidase treatment are shown by Coomassie gel staining.

FIG. 2A-H shows gpl20 glycopeptide epitopes are recognized by CD4+ T cells. B ALB/c mice were immunized with pooled gpl20 gly copeptides (prepared by protease digestion of gpl20). After booster immunization, CD4+ T cells were isolated and stimulated in vitro with either intact gpl20 or PNGase F-treated, deglycosylated gpl20 (DG-gpl20) in the presence of mitomycin C-treated APCs for five days. (FIG. 2A, B) Flow cytometric analysis of T cell proliferation by CFSE division among CD4+ T cells. (FIG. 2C) Recognition of coated gpl20 by antiserum from mice immunized with gpl20 glycopeptides in the presence of the indicated inhibitors was examined by inhibition ELISA. Intact gpl20, but not deglycosylated gpl20, blocked recognition in a dose-dependent manner. Serum titers are reported as OD at 405 nm. (FIG. 2D, E) Production of IL-4 (FIG. 2D) and IFN-g (FIG. 2E) in the culture supernatant after T cell stimulation was measured by ELISA. (FIG. 2F-H) Serum from mice immunized with pooled gpl20 glycopeptides were collected 7 days after booster immunization. Titers of IgGl (FIG. 2F), IgG2a (FIG. 2G), and IgG3 (FIG. 2H) for recognition of glycosylated gpl20 or deglycosylated gpl20 were measured by ELISA n > 4 mice per group.

FIG. 3A-E shows characterization of a gpl20 glycotope presented by MHCII. (FIG. 3A- C) MS/MS spectra of a gpl20 variable-region (V2) glycopeptide (designated GpepIP) identified from MHCII-bound epitopes shows b- and y-ions defining peptide sequence KLDVVPID N187TSY in the non-protease group (FIG. 3 A), peptide sequence LDVVPIDNNN187TSYR in the trypsin-treated group (FIG. 3B), peptide sequence LDVVPID N187TSYR in the chymotrypsin-treated group (FIG. 3C), and the N-glycosylation site atN187 in all three groups. The @ symbol denotes that the conversion of an Asn residue to Asp with a heavy oxygen was detected at the indicated position. (FIG. 3D, E) Binding of the GpepIP epitope to MHCII molecule was verified. (FIG. 3D) Purified MHCII monomers (mouse allele I- Ad) were loaded with an equal amount of the indicated peptides. Peptide-loaded MHCII heterodimers were detected by running of complexes in IEF gel and immunoblotting with mouse MHCII antibody. (FIG. 3E) Bands corresponding to GpepIP/MHCII, pepIP/MHCII, and OVA peptide/MHCII complexes were excised from IEF gels and subjected to LC-MS/MS analysis. Extracted ion chromatograms demonstrate the binding of three GpepIP glycoforms (M5N2 being the most abundant). PepIP and the positive control (OVA peptide) also bound and stabilized MHCII heterodimers.

FIG. 4 shows glycopeptide profile of the GpepIP (LDVVPIDNISIN187TSYR) region in gpl20 expressed in 293-F cells, as analyzed by MS. The original full MS spectrum (averaged spectrum) was obtained from 30 min to 40min of the LC-MS data, with the target glycopeptides eluted during the LC-MS run. The spectrum presented here shows data deconvoluted with Xtract software for quantification purposes.

FIG. 5A-C shows characterization of three GpepIP variants. (FIG. 5A) GpepIP was recombinantly expressed in either Free Sty leTM 293-F cells or N-acetylglucosaminyltransferase I mutant (GnTI-/-) cells. A peptide variant without glycan modification (pepIP) was chemically synthesized. The molecular weights of the three glycopeptides were determined by SDSPAGE and silver staining. (FIG. 5B) GpepIP expressed in GnTI-/- cells was treated with PNGase F to remove N-glycans. (FIG. 5C) Glycosylation of GpepIP expressed in GnTI-/- cells and pepIP was detected by western blotting with the lectin ConA.

FIG. 6A-B shows Glycopeptide profile of GpepIP expressed in GNTI-/- cells analyzed by MS. (FIG. 6A) The deconvoluted MS spectrum was obtained from the LC-MS time region 14.23-20.48 min. The peptide regions KLDVVPIDNNNTSY and KLD V VPIDNNNT S (missing one amino acid at the C terminal) with His tagging (5 or 6 histidines) were identified. (FIG. 6B) Gly copeptides with the sequence HHHHHKLDVVPIDNNNTS were found between 13 min and 18 min in the LC-MS run. The deconvoluted spectrum from this time region is displayed. The major gly can observed is Hex5HexNAc2; Hex4HexNAc2, Hex5HexNAc2Fuc2, and Hex4HexNAc2Fuc2 were minor components. No complex-type structure or sialylated structure was detected.

FIG. 7 shows full MS profile of N-glycans released from GpepIP expressed in 293-F cells. The glycopeptides from 293-F cells were eluted between 20 and 50 min in the LC-MS run, and the deconvoluted MS spectrum was obtained from this time region. Glycan composition of this glycopeptide showed that the recombinant glycopeptide carries more complex-type glycans with heavy fucosylation and sialylation, while high-mannose types were detected as minor components.

FIG. 8A-J shows the glycopeptide epitope GpepIP elicits a glycan-specific cellular and humoral immune response. (FIG. 8A, B) CD4+ T cells obtained from mice immunized with gpl20 were stimulated in vitro with either GpepIP or pepIP, and T cell proliferation was examined by flow cytometry with use of CFSE fluorescence dilution through cell division. (FIG. 8C, D) CD4+ T cells obtained from mice immunized with GpepIP expressed in GnTI-/- cells (FIG. 8C) or with pepIP (FIG. 8D) were stimulated with GpepIP expressed in GnTI-/- or 293F cells or with pepIP, and T cell proliferation was examined by CFSE flow cytometry'. (FIG. 8E, F) Antisera from mice immunized with GpepIP or pepIP were titrated for IgG binding to immobilized GpepIP (FIG. 8E) or pepIP (FIG. 8F) by ELISA. (FIG. 8G) Antisera from GpepIP and pepIP immunization groups recognize gpl20 expressed in 293-F and GnTI-/- cells differentially as measured by ELISA. (FIG. 8H-J) Serum from mice immunized with GpepIP expressed in GnTI-/- cells were collected 7 days after booster immunization. Titers of IgGl (FIG. 8H), IgG2a (FIG. 81), and IgG3 (FIG. 8J) for recognition of glycosylated gpl20 or deglycosylated gpl20 were measured by ELISA. FIG. 9A-F shows transcriptomic analysis of GpepIP- and pepIP-stimulated CD4+ T cell populations. (FIG. 9A) Gating strategy of sorting antigen specific CD4+ T cell populations by flow cytometry is shown. Three weeks after the third immunization with GpepIP or pepIP, splenic and lymph node cells were isolated and stimulated in vitro with GpepIP or pepIP for three days. CD4+CD69+ and CD4+CD69- T cell populations were then sorted by flow cytometry. (FIG. 9B) Dendrogram and hierarchical clustering heat map of genes from control, GpepIP and pepIP populations. The blue and red bands indicate low and high gene expression quantity, respectively. The vertical distances between branches of the dendrogram represent the similarity of gene expression profiles between samples. Biological replicates showed the highest degree of correlation followed by GpepIP or pepIP stimulated populations. (FIG. 9C) The number of upregulated, downregulated and unchanged genes in GpepIP and pepIP stimulated CD4+ T cells compared to control or to each other is shown. (FIG. 9D) KEGG pathway enrichment analysis of DEGs from each comparison. Point size indicates DEG number (The bigger dots refer to larger amount). Rich Factor refers to the value of enrichment factor which is the quotient of the number of DEGs and total gene amount in that pathway. Pathways associated with Th cell differentiation were highlighted by red rectangles. (FIG. 9E) Heat map depicts the DEGs associated with Thl and Th2 cell differentiation, IL-17 signaling pathway and Thl7 cell differentiation between GpepIP and pepIP specific CD4+ T cells with normalization to control. Heatmap colors represent the log2 fold change values relative to the control. (FIG. 9F) Volcano plot showing the gene signature of GpepIP compared to pepIP specific CD4+ T cells. X axis represents log2 transformed fold change. Y axis represents -log 10 transformed significance. Red points represent upregulated DEGs. Blue points represent down-regulated DEGs. Gray points represent non- DEGs. Genes associated with Th cell differentiation were labeled and highlighted.

FIG. 10A-B shows GO analysis and gene expression signature of GpepIP specific CD4+ T cells. (FIG. 10A) The most enriched GO of DEGs in GpepIP specific CD4+ T cells compared to control cells was represented. The number of genes of GO in biological process, cellular component and molecular function were shown. (FIG. 10B) Volcano plot showing the gene signature of GpepIP specific CD4+ T cells compared control. DEGs (greater than twofold; P value < 0.05) were shown as colored dots. Genes associated with Thl and Th2 signaling, Thl7 signaling and activated T cell co-stimulatory molecules were labeled. FIG. 11 A-E shows cytokine profile of GpepIP and pepIP stimulation. Splenic and lymph node cells isolated from GpepIP or pepIP immunized mice were stimulated with GpepIP or pepIP for 5 days. Th-cell related cytokines in the supernatants from GpepIP (FIG. 11 A) or pepIP (FIG. 1 IB) stimulation compared to no stimulation (medium) were analyzed by a multiplex- based assay. (FIG. 11C) Production of cytokines associated with Th2 (IL4, IL-5, IL-6, IL-10 and IL-13) and Thl7 (IL-17A, IL-17F and IL-22) was examined in GpepIP- and pepIP-stimulated groups. (FIG. 1 ID, E) Cells in (FIG. 11A) and (FIG. 1 IB) were stimulated with GpepIP or pepIP or in medium for 3 days. Cytokines IFN-g, IL-5 and IL-17A on CD4+ T cells were assessed by intracellular cytokine staining and flow cytometry.

FIG. 12A-B shows Th-cell related cytokine analysis of GpepIP- or pepIP-stimulated CD4+ T cells. Splenic and lymph node cells isolated from GpepIP or pepIP immunized mice were stimulated with GpepIP or pepIP for 5 days. Cytokines IFN-g, IL-5 and IL-17A on CD4+ T cells were assessed by intracellular cytokine staining and flow cytometry.

FIG. 13A-G shows GpepIP specific CD4+ T cells exhibit high potency on helping humoral immune responses to HIV trimer. (FIG. 13A) Immunization scheme. BALB/c mice were primed twice by subcutaneous injection of GpepIP or pepIP emulsified in Freund’s adjuvant or of adjuvant alone. Three weeks later, all groups were immunized with the clade A BG505 gpl40 NFL trimer emulsified in incomplete Freund’s adjuvant. Sera were collected at the indicated time points. Mice were euthanized 10 days after trimer immunization. (FIG. 13B) Splenic and lymph node cells were isolated and stimulated with GpepIP or pepIP in vitro for five days. T cell proliferation by CFSE dilution was measured by flow cytometry. GpepIP and pepIP specific CD4+ T cells were enriched in GpepIP and pepIP primed mice, respectively. (FIG. 13C) BG505- specific IgG production was examined in all three groups across the indicated time points by ELISA. Antibody titers were significantly higher in mice primed with GpepIP than in those primed with pepIP or adjuvant alone. (FIG. 13D) GC response, defined by the percentage of GL7+Fas+ B cells, was evaluated in all three groups on day 10 after trimer immunization by flow cytometry. (FIG. f 3E-G) Expression levels of activation marker CD69 (FIG. 13E), CD80 (FIG. 13F) and MHCII (FIG. 13G) were detected on splenic B cells after in vitro stimulation with the BG505 trimer for 3 days. MFI, mean fluorescence intensity.

FIG. 14A-E shows GpepIP primary immunization prior to trimer immunization elicits functional antibody production. (FIG. 14A) Immunization scheme. BALB/c mice were primed three times (with a 3-week interval) by subcutaneous injection of GpepIP or pepIP emulsified in Freund’s adjuvant or of adjuvant alone. Subsequently, all groups were immunized with the clade A BG505 gpl40 NFL trimer adjuvanted with Alum for three times (with a 3-week interval). Sera were collected 7 days after each trimer immunization (Post 1, 2 and 3). (FIG. 14B) BG505- specific IgG production was examined in all three groups post each trimer immunization by ELISA. (FIG. 14C) Antisera from all three groups after the third trimer immunizations were analyzed for IgG subclass switching by ELISA. (FIG. 14D) The neutralizing activity (neutralization 50% inhibitory dilution (ID50)) of antisera from adjuvant, GpepIP and pepIP primary followed by three BG505 booster immunizations were tested against tier 1 and tier 2 HIV-1 viruses via a TZM-bl cell-based neutralization assays. MLV-pseudotyped virus was used as negative control for non-HIV-specific inhibitory activity in the assay. Antibody CHOI-31 was used as positive control (shown as antibody concentration). (FIG. 14E) BG505 was chemically labeled with fluorescein isothiocyanate (FITC) and incubated with BMDCs at 37°C for two hours. Cells were then collected and antigen uptake was measured by flow cytometry. To evaluate antisera for their function, fluorophore-labeled BG505 was pre-incubated with antisera used in (FIG. 14E) at different dilutions before adding into BMDCs. The uptake rate is defined as FITC positive cells compared to no BG505 (medium) group.

FIG. 15 shows trimer-specific IgG titers. BG505-specific IgG production was examined in antisera from adjuvant, GpepIP and pepIP primed and post the third trimer boost by ELISA. All three groups have equivalent BG505 IgG titers.

FIG. 16A-B shows human CD4+ T cells are stimulated by gpl20 glycopeptide epitopes. PBMCs from an HIV- 1 -infected donor and an HIV- 1 -negative healthy donor were stimulated in vitro with pooled gpl20 glycopeptides and PNGase F-treated, deglycosylated glycopeptide pool or GpepIP and pepIP for three days. Production of IFN-g (FIG. 16A) and TNF-a (FIG. 16B) in the culture supernatant after T cell stimulation was measured by ELISA.

The schematic drawings are not necessarily to scale. Like numbers used in the figures refer to like components, steps and the like. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number. In addition, the use of different numbers to refer to components is not intended to indicate that the different numbered components cannot be the same or similar to other numbered components. DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Glycopeptides

The present disclosure provides isolated glycopeptides. As used herein, the term “protein” refers broadly to a polymer of two or more amino acids joined together by peptide bonds. The term “protein” also includes molecules which contain more than one protein joined by a disulfide bond, or complexes of proteins that are joined together, covalently or noncovalently, as multimers (e.g., dimers, tetramers). Thus, the terms peptide, oligopeptide, and polypeptide are all included within the definition of protein and these terms are used interchangeably. The term “glycopeptide” includes any molecule that contains both a protein component and at least one carbohydrate component. As used herein, the term glycoprotein is inclusive of a glycoprotein, a glycopolypeptide and a proteoglycan. An “isolated” compound, such as a polynucleotide or glycopeptide, is one that has been removed from its natural environment. Polynucleotides and glycopeptides that are produced by recombinant, enzymatic, or chemical techniques are considered to be isolated and purified by definition, since they were never present in a natural environment.

A glycopeptide of the present disclosure includes an amino acid sequence that includes a series of consecutive amino acids derived from the amino acid sequence of a human HIV-1 gpl20 protein, is glycosylated with at least one glycosylation site and at least one linked glycan as described herein, and binds a class 2 major histocompatibility complex in such a way that it is presented to a T cell receptor, and/or induces the production of antigen-specific antibodies, and/or induces the production of T cells that are specifically stimulated by the glycopeptide

Protein component

A glycopeptide described herein includes consecutive amino acids of a human HIV-1 gpl20 protein. Examples include, but are not limited to, those sequences in Table 1 of Example 1 shown as having an N-glycosylation site. While the cysteine residues of the glycopeptides shown in Table 1 are carbamidomethylated, in one embodiment cysteine residues of a glycopeptide of the present disclosure are not carbamidomethylated. In one embodiment, a glycopeptide described herein includes at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive amino acids of a human HIV-1 gpl20 protein. In one embodiment, a glycopeptide described herein includes no greater than 20, no greater than 19, no greater than 18, no greater than 17, no greater than 16, no greater than 15, no greater than 14, no greater than 13, no greater than 12, no greater than 11, no greater than 10, no greater than 9, no greater than 8, or no greater than 7consecutive amino acids of a human HIV-1 gpl20 protein.

Without intending to be limiting, one exemplary glycopeptide can include an amino acid sequence LDVVPIDNNNTSY (SEQ ID NO:l), where the asparagine at position 10 is glycosylated. The glycopeptide depicted at SEQ ID NO: 1 corresponds to amino acids 178-190 of a gpl20 molecule of HIV-1. This sequence is derived from a GP120 of HIV-1 clade B. Other examples of useful glycopeptides can be based on the GP120 of the other HIV-1 clades, such as clades A, B and C. In one embodiment, the amino acid sequence of SEQ ID NO:l includes a lysine at the amino-terminal end, an arginine at the carboxy-terminal end, or both a lysine at the amino-terminal end and an arginine at the carboxy-terminal end.

Also included in the present disclosure are glycopeptides that include a portion of SEQ ID NO: 1 or another glycopeptide described herein. For instance, a glycopeptide can include at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or 14 consecutive amino acids from SEQ ID NO:l.

In one embodiment, a glycopeptide described herein includes additional amino acids that are normally or naturally found flanking the sequence depicted at, for instance, SEQ ID NO:l, in a natural gpl20 molecule of HIV-1. In one embodiment, the natural gpl20 molecule is SEQ ID NO: 10 (encoded by reagent pSyngpl20JR-FL, NIH AIDS Reagent Program). The additional amino acids do not include a naturally occurring full length gpl20 molecule. The additional amino acids can be at the amino terminal end, the carboxy terminal end, or both.

Other examples of glycopeptides of the present disclosure include those having structural similarity with the amino acid sequence of SEQ ID NOT or another glycopeptide described herein. As used herein, a glycopeptide may be “structurally similar” to a reference glycopeptide if the amino acid sequence of the glycopeptide possesses a specified amount of structural similarity and/or structural identity compared to the reference glycopeptide. Thus, a glycopeptide may have structural similarity to a reference glycopeptide if, compared to the reference glycopeptide, it possesses a sufficient level of amino acid structural identity, amino acid structural similarity, or a combination thereof. A glycopeptide can be isolated from a cell or from an MHC II complex or can be produced using recombinant techniques, or chemically or enzymatically synthesized using routine methods. Methods for determining whether a protein has structural similarity with the amino acid sequence of SEQ ID NO: 1 are described herein.

The amino acid sequence of a gly copeptide having structural similarity to SEQ ID NO: 1 or another glycopeptide described herein can include conservative substitutions of amino acids present in SEQ ID NO:l or another glycopeptide described herein. A conservative substitution is typically the substitution of one amino acid for another that is a member of the same class. For example, it is well known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity, and/or hydrophilicity) may generally be substituted for another amino acid without substantially altering the secondary and/or tertiary structure of a polypeptide. For the purposes of this invention, conservative amino acid substitutions are defined to result from exchange of amino acids residues from within one of the following classes of residues: Class I: Gly, Ala, Val, Leu, and He (representing aliphatic side chains); Class II: Gly, Ala, Val, Leu, lie, Ser, and Thr (representing aliphatic and aliphatic hydroxyl side chains); Class III: Tyr, Ser, and Thr (representing hydroxyl side chains); Class IV: Cys and Met (representing sulfur-containing side chains); Class V: Glu, Asp, Asn and Gin (carboxyl or amide group containing side chains); Class VI: His, Arg and Lys (representing basic side chains); Class VII: Gly, Ala, Pro, Trp, Tyr, lie, Val, Leu, Phe and Met (representing hydrophobic side chains); Class VIII: Phe, Trp, and Tyr (representing aromatic side chains); and Class IX: Asn and Gin (representing amide side chains). The classes are not limited to naturally occurring amino acids, but also include artificial amino acids, such as beta or gamma amino acids and those containing non-natural side chains, and/or other similar monomers such as hydroxyacids.

Whether a glycopeptide is structurally similar to a protein of SEQ ID NO: 1 can be determined by aligning the residues of the two proteins (for example, a candidate protein and any appropriate reference protein described herein) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. A reference protein may be a protein described herein. In one embodiment, a reference protein is a protein described at SEQ ID NO: 1 or another glycopeptide described herein. A candidate protein is the protein being compared to the reference protein. A candidate protein can be produced using recombinant techniques, or chemically or enzymatically synthesized.

Unless modified as otherwise described herein, a pair-wise comparison analysis of amino acid sequences can be carried out using the Blastp program of the Blastp suite-2sequences search algorithm, as described by Tatusova et ah, (FEMS Microbiol Lett , 174, 247-250 (1999)), and available on the National Center for Biotechnology Information (NCBI) website. The default values for all blastp suite-2sequences search parameters may be used, including general paramters: expect threshold=10, word size=3, short queries=on; scoring parameters: matrix = BLOSUM62, gap costs=existence:ll extension: 1, compositional adjustments=conditional compositional score matrix adjustment. Alternatively, proteins may be compared using other commercially available algorithms, such as the BESTFIT algorithm in the GCG package (version 10.2, Madison WI).

In the comparison of two amino acid sequences, structural similarity may be referred to by percent “identity” or may be referred to by percent “similarity.” “Identity” refers to the presence of identical amino acids. “Similarity” refers to the presence of not only identical amino acids but also the presence of conservative substitutions.

In one embodiment, the amino acid sequence of a glycopeptide having structural similarity to SEQ ID NO: 1 or another glycopeptide described herein can include at least 1, at least 2, at least 3, at least 4, or at least 5 conservative substitutions of amino acids present in SEQ ID NO: 1 or another glycopeptide described herein. In one embodiment, the amino acid sequence of a glycopeptide having structural similarity to SEQ ID NO: 1 or another glycopeptide described herein can include no greater than 5, no greater than 4, no greater than 3, no greater than 2, or no greater than 1 conservative substitutions of amino acids present in SEQ ID NO:l or another glycopeptide described herein.

Thus, as used herein, reference to an amino acid sequence disclosed at SEQ ID NO:l or another glycopeptide described herein can include a protein with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence similarity to the reference amino acid sequence. Alternatively, as used herein, reference to an amino acid sequence disclosed at SEQ ID NO: 1 or another glycopeptide described herein can include a protein with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least

82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least

89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least

96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the reference amino acid sequence.

Unless a specific level of sequence similarity and/or identity is expressly indicated herein (e.g., at least 80% sequence similarity, at least 90% sequence identity, etc.), reference to the amino acid sequence of an identified SEQ ID NO includes variants having sequence similarity or sequence identity of at least 80%.

A glycopeptide having structural similarity to a glycopeptide described herein has immunogenic activity. Thus, a glycopeptide having one or more conservative and/or nonconservative substitutions compared to a glycopeptide described herein has immunogenic activity.

A glycopeptide described herein can be a fusion protein, where the additional amino acids can be heterologous amino acids. As used herein, “heterologous amino acids” refers to amino acids that are not normally or naturally found flanking the sequence depicted at, for instance, SEQ ID NO: 1, in a natural gpl20 molecule of HIV-1. In one embodiment, the natural gpl20 molecule is SEQ ID NO: 10 (encoded by reagent pSyngpl20JR-FL, NET AIDS Reagent Program). For instance, the additional amino acid sequence may be useful for purification of the fusion protein by affinity chromatography. Various methods are available for the addition of such affinity purification moieties to proteins. Representative examples may be found in Hopp et al. (U.S. Pat. No. 4,703,004), Hopp et al. (U.S. Pat. No. 4,782,137), Sgarlato (U.S. Pat. No. 5,935,824), and Sharma Sgarlato (U.S. Pat. No. 5,594,115).

In one embodiment a fusion protein is a series of two or more glycopeptides described herein covalently joined together as a multimer. The number of glycopeptides joined together is not limiting and in one embodiment there is no upper number of glycopeptides that can be present in a multimer. In one embodiment, the number of multimers can be at least 2, at least 5, at least 10, at least 50, or at least 100. In one embodiment, the glycopeptides of a multimer are joined as a fusion protein where the carboxy -terminal end of one glycopeptide is attached to the amino-terminal end of the next one. In some embodiments, a multimer includes a spacer, e.g., one or more amino acids between the glycopeptides of a multimer. A spacer is an amino acid sequence that joins protein domains in a fusion protein. A spacer can be flexible or rigid, and in one embodiment is flexible. In one embodiment, a spacer can be at least 3, at least 4, at least 5, or at least 6 amino acids in length. It is expected that there is no upper limit on the length of a linker used in a multimer described herein; however, in one embodiment, a spacer is no greater than 10, no greater than 9, no greater than 8, or no greater than 7 amino acids in length. A spacer sequence can be any amino acid sequence, and can have small R groups to reduce steric hindrance.

In one embodiment, a spacer includes a cleavable moiety, e.g., an amino acid sequence that is recognized and cleaved by an enzyme such as a protease, or a chemical moiety that is acid labile. A number of such cleavable moieties are known in the art. For example, cleavable sequences can include those recognized by cathepsins (Conus and Simon, 2008, Biochem. Pharmacol., 76:1374-1382; Arnold et al., 1997, Eur: J. Biochm. 249:171-179; Roberts, 2005, Drug News Perspect, 18(10):605; and Plugeret al., 2002, Eur: J. Immunol. 32:467-476). In some embodiments, the cleavable sequence is a recognition sequence for a protease present in an endosome. Acid labile moieties are also known in the art, and can include 4-(4-Hydroxymethyl- 3-methoxyphenoxy)butyric acid (SIGMA) (Riniker et al., 1993, Tetrahedron (49)41:9307-9320).

Carbohydrate component

The carbohydrate component of a glycopeptide is commonly referred to as a “glycan.” A glycan may contain one monosaccharide, or it may contain two or more monosaccharides linked by glycosidic bonds. A glycan can include nonrepeating or repeating monosaccharides, or both.

As used herein, the term “glycan” is interchangeable with the term saccharide, which includes a monosaccharide, a disaccharide, a trisaccharide, etc.; it can include an oligosaccharide or a polysaccharide. An oligosaccharide is an oligomeric saccharide that contains two or more saccharides. The structure of an oligosaccharide is typically characterized by a particular identity, order, linkage positions (including branch points), and linkage stereochemistry (a, b) of the monomers, and as a result has a defined molecular weight and composition. In a polysaccharide, the identity, order, linkage positions (including branch points) and/or linkage stereochemistry can vary from molecule to molecule. “Glycosylation” refers to the covalent attachment of at least one saccharide moiety to a molecule. Glycosidic linkages include O- glycosidic linkages, N-glycosidic linkages, S-glycosidic linkages and C-glycosidic linkages. An O-glycosidic linkage is formed between the anomeric carbon (Cl) of a saccharide and an oxygen atom of another molecule, while an A-glycosidic linkage is formed between the anomeric carbon (Cl) of a saccharide and a nitrogen atom of another molecule. Likewise, S-glycosidic linkages and C-glycosidic linkages involve a sulphur and carbon atom from another molecule, respectively. Thus, the glycan component of a glycopeptide can be A-l inked, G-l inked, or S- linked to the protein component of the glycopeptide. In addition, glycosidic linkages are classified according to the ring position of the carbon atoms participating in the bond. For example, a 1,4 glycosidic linkage is formed between the first carbon (Cl) on a first saccharide and the fourth carbon (C4) on a second saccharide while a 1,6 glycosidic linkage is formed between the first carbon (Cl) on a first saccharide and the sixth carbon (C6) on a second saccharide. Glycosidic linkages are further classified as a-glycosidic or b-glycosidic according to whether the substituent groups on the carbons flanking the oxygen in the saccharide are pointing in the same or opposite directions. The term “glycosylation” as used herein should be broadly construed so as to encompass the covalent linkage of any other carbohydrate moieties such as mannose, and as such includes mannosylation.

A glycan can be branched or unbranched. A complex glycan is a glycan that contains at least one branch point. In a complex or branched glycan, the monosaccharide at the branch point is covalently linked to two other saccharides at carbons other than Cl. For example, a branch point monosaccharide may be linked to other monosaccharides at C4 and C6, in addition to being linked to another monosaccharide or to an amino acid at Cl. A complex glycan may be, without limitation, biantennary, triantennary, or tetraantennary.

A glycopeptide described herein includes at least one glycan attached to the protein component. In one embodiment, a glycopeptide includes at least 1, at least 2, at least 3, or at least 4 glycans. In one embodiment, a glycopeptide includes no greater than 4, no greater than 3, no greater than 2, or no greater than 1 glycans. In one embodiment, the glycan is an oligo mannose structure. As used herein, the term “oligo mannose structure” refers to an oligosaccharide that includes mannose residues. An “oligo mannose structure” includes a common structure, referred to as a core, which typically contains two N-acetyl glucosamine residues and three or more mannose residues. Terminal modifications and core modifications of a glycan can include glycosylations. Core glycosylation includes the addition of one or more glycosyl moieties to a core N-acetylglucosamine and addition of one or more glycosyl moieties to a core mannose. In one embodiment, core glycosylation includes the addition of one or more glycosyl moieties to one or more core mannose or N-acetylglucosamine residues. In one embodiment, one or more mannose residues is attached to one or more core mannose residues.

An oligo mannose structure includes at least 3, at least 4, at least 5, at least 6, or at least 7 mannose residues. In one embodiment, an oligo mannose residue includes no greater than 9, no greater than 8, no greater than 7, no greater than 6, no greater than 5, no greater than 4, or no greater than 3 mannose residues. An oligo mannose structure typically terminates in mannose residues at the non-reducing end of the glycan. One or more glycans of a glycopeptide may contain modifications such as sulfation or phosphorylation. In one embodiment, a glycan of a glycopeptide may be a complex type N-glycan or a hybrid N-glycan. Examples of glycopeptides include the molecules shown in Figure 4 having 2 N-acetylglucosamine residues (N2) and 5, 6, or 7 mannose residues (M5, M6, and M7, respectively) and the molecules shown in Figure 4F having the structures M4N2 and M5N2. In one embodiment, a processed glycan after endosomal processing of the glycopeptide contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 saccharide monomers. In one embodiment, a processed glycan after endosomal processing of the glycopeptide contains no greater than 9, no greater than 8, no greater than 7, no greater than 6, no greater than 5, no greater than 4, no greater than 3, no greater than 2, or no greater than 1 saccharide monomers.

Activity

A glycopeptide described herein has immunogenic activity (IA). IA includes ability to bind to a MHC II molecule, and/or induce the production of antigen-specific antibodies, and/or induce the production of T cells that are specifically stimulated by the glycopeptide.

Whether a protein has IA can be determined by in vitro or in vivo assays. In one embodiment, an assay determines glycopeptide binding to MHCII molecules, such a a mouse or a human MHCII molecule, and can be carried out as described in Example 1. A glycopeptide described herein will bind to, and be presented by, MHC II molecules. In another embodiment, an assay determines the T cell response to glycopeptides and can be carried out as described in Example 1. When a gly copeptide described herein is used to immunize a subject, T cells from the subject will be stimulated by the gly copeptide to produce interleukin-4 and interferon- gamma. In another embodiment, an assay determines the production of antigen-specific antibodies and can be carried out as described in Example 1. The antigen-specific antibodies can react with the gly copeptide used to immunize the subject, or react with a second antigen administered to the subject with the gly copeptide or following immunization of the subject with the glycopeptide. Typically, the second antigen is one that shares at least one epitope with the glycopeptide. An example of such a protein is one based on an HIV gpl40, such as a trimer envelope protein.

Polynucleotides

The present disclosure also includes isolated polynucleotides encoding a protein described herein. A polynucleotide encoding a protein described herein can have a nucleotide sequence encoding a protein having the amino acid sequence shown in, e.g., SEQ ID NO: 1 or a glycopeptide disclosed herein, or a protein that is structurally similar. A nucleotide sequence of a polynucleotide encoding a protein described herein can be readily determined by one skilled in the art by reference to the standard genetic code, where different nucleotide triplets (codons) are known to encode a specific amino acid. As is readily apparent to a skilled person, the class of nucleotide sequences that encode any protein described herein is large as a result of the degeneracy of the genetic code, but it is also finite.

A polynucleotide encoding a glycopeptide described herein can be present in a vector. A vector is a replicating polynucleotide, such as a plasmid, phage, or cosmid, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide. Construction of vectors containing a polynucleotide employs standard ligation techniques known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press (1989). A vector may provide for further cloning (amplification of the polynucleotide), i.e., a cloning vector, or for expression of the polynucleotide, i.e., an expression vector. The term vector includes, but is not limited to, plasmid vectors, viral vectors, cosmid vectors, and artificial chromosome vectors. Examples of viral vectors include, for instance, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, and herpes virus vectors. Typically, a vector is capable of replication in a microbial host, for instance, a prokaryotic bacterium, such as E. coli. Preferably the vector is a plasmid.

Selection of a vector depends upon a variety of desired characteristics in the resulting construct, such as a selection marker, vector replication rate, and the like. Suitable host cells for cloning or expressing the vectors herein include eukaryotic cells. Suitable eukaryotic cells include, but are not limited to, human Embryonic Kidney (HEK) 293 cells and Chinese hamster ovary (CHO) cells. Vectors may be introduced into a host cell using methods that are known and used routinely by the skilled person. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells.

Polynucleotides can be produced in vitro or in vivo. For instance, methods for in vitro synthesis include, but are not limited to, chemical synthesis with a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic polynucleotides and reagents for such synthesis are well known.

An expression vector optionally includes regulatory sequences operably linked to the coding region. The disclosure is not limited by the use of any particular promoter, and a wide variety of promoters are known. Promoters act as regulatory signals that bind RNA polymerase in a cell to initiate transcription of a downstream (3' direction) coding region. The promoter used may be a constitutive or an inducible promoter. It may be, but need not be, heterologous with respect to the host cell.

An expression vector may optionally include a ribosome binding site and a start site (e.g., the codon ATG) to initiate translation of the transcribed message to produce the polypeptide. It may also include a termination sequence to end translation. The polynucleotide used to transform the host cell may optionally further include a transcription termination sequence.

A vector introduced into a host cell optionally includes one or more marker sequences, which typically encode a molecule that inactivates or otherwise detects or is detected by a compound in the growth medium. For example, the inclusion of a marker sequence may render the transformed cell resistant to an antibiotic, or it may confer compound-specific metabolism on the transformed cell. Examples of a marker sequence are sequences that confer resistance to kanamycin, ampicillin, chloramphenicol, tetracycline, and neomycin. Proteins described herein may be produced using recombinant DNA techniques, such as an expression vector present in a cell (e.g., a genetically modified cell described herein). Such methods are routine and known in the art. A glycopeptide can also be synthesized in vitro , e.g., by solid phase peptide synthetic methods. The solid phase peptide synthetic methods are routine and known in the art. A glycopeptide produced using recombinant techniques or by solid phase peptide synthetic methods may be further purified by routine methods, such as fractionation on immunoaffmity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on an anion-exchange resin such as DEAE, chromatofocusing, SDS- PAGE, ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, or ligand affinity.

Genetically modified cells

The present disclosure also includes genetically modified cells that have an exogenous polynucleotide encoding a glycosylated protein described herein. As used herein, “genetically modified cell” refers to a cell into which has been introduced an exogenous polynucleotide. For example, a cell is a genetically modified cell by virtue of introduction into a suitable cell of an exogenous polynucleotide. As used herein, the term “exogenous” refers to a compound, such as a polynucleotide or glycopeptide, that is not normally or naturally found in a specific cell. Compared to a control cell that is not genetically modified, a genetically modified cell can exhibit production of a glycopeptide described herein. A polynucleotide encoding a glycopeptide can be present in the organism as a vector or integrated into a chromosome. A genetically engineered cell can stably express a glycopeptide, or the expression can be transient.

Examples of cells include, for instance, prokaryotic (e.g., microbial) and eukaryotic. Examples of eukaryotic cells include yeast, insect, and animal cells. Examples of animal cells include vertebrate cells, such as mammalian cells. An example of a mammalian cell includes, but is not limited to, HEK293 N-acetylglucosaminyltransferase I (GnTI)-deficient cells. An animal cell can be an in vitro cell (e.g., a cell that is capable of long term culture in tissue culture medium), or an ex vivo cell (e.g., a cell that has been removed from the body of a subject and capable of limited growth in tissue culture medium).

In one embodiment, a genetically modified cell useful herein is able to glycosylate proteins to include oligomannose N-glycans but prevent the production of complex N-glycans. In one embodiment, a suitable cell is one that includes a mutation in N- acetylglucosaminyltransferase I (GnTI), an enzyme used for the conversion of oligomannose N- glycans to complex N-glycans. An example of a cell having a mutation in N- acetylglucosaminyltransferase I (GnTI-/-) is HEK293S GnTT, available from the ATCC® (Manassas, VA) as CRL-3022™. Alternatively, to generate gly copeptides with heterogeneity in glycan composition, Kifunensine, a mannosidase inhibitor of the ER and Golgi mannosidase I, can be added during expression in 293-F cells to produce glycopeptides.

Compositions

Also provided are compositions that include a glycopeptide described herein or a polynucleotide encoding a glycopeptide. The glycopeptide present in a composition can be a monomer (e.g., one glycopeptide) or a multimer (e.g., at least two glycopeptides joined as a fusion protein), or a combination thereof. For instance, a composition can include, but is not limited to, a mixture of different multimers, a mixture of a monomer and different multimers, or a mixture of different monomers. In one embodiment, the glycan or gly cans attached to the glycopeptide - whether the glycopeptide is a monomer or multimer - of a composition is the same (e.g., the oligosaccharide is M5N2), and in another embodiment the oligosaccharides attached vary (e.g., the oligosaccharides can be M5N2 and M4N2 or other glycopeptides as endolysososomal processing products ).

A composition can include a pharmaceutically acceptable carrier. As used herein “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

A composition may be prepared by methods well known in the art of pharmaceutics. In general, a composition can be formulated to be compatible with its intended route of administration. Administration may be systemic or local. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular), enteral (e.g., oral), and topical (e.g., epicutaneous, inhalational, transmucosal) administration. Appropriate dosage forms for enteral administration of the compound of the present disclosure include, but are not limited to, tablets, capsules, or liquids. Appropriate dosage forms for parenteral administration may include intravenous or intraperitoneal administration. Appropriate dosage forms for topical administration include, but are not limited to, nasal sprays, metered dose inhalers, dry-powder inhalers, or by nebulization.

Solutions or suspensions can include the following components: a sterile diluent such as water for administration, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; electrolytes, such as sodium ion, chloride ion, potassium ion, calcium ion, and magnesium ion, and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.

Compositions can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile solutions or dispersions. For parenteral administration, suitable carriers include physiological saline, bacteriostatic water, phosphate buffered saline (PBS), and the like. A composition is typically sterile and, when suitable for injectable use, should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile solutions can be prepared by incorporating the active compound (e.g., a glycopeptide described herein) in the required amount in an appropriate solvent with one or a combination of ingredients routinely used in pharmaceutical compositions, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and any other appropriate ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterilized solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier. Pharmaceutically compatible binding agents and/or other useful materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation (e.g., topical administration), the active compounds can be delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and/or fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The active compounds may be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. In one embodiment, a composition can also include another compound useful in eliciting an immune response. In one embodiment, a useful compound is one the elicits an immune response to HIV. One such compound includes a stabilized envelope trimer. Stabilized envelope trimers are available as immunogens (Sanders, et al., 2013, PLoS pathogens, 9, el 003618, doi:10.1371/journal.ppat.l003618).

Toxicity and therapeutic efficacy of the active compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Recombinant glycopeptides exhibiting high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration used. For a compound used in the methods described herein, the therapeutically effective dose can be estimated initially from animal models. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of signs of disease). Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured using routine methods.

A composition is administered in an amount sufficient to provide an immunological response to a glycopeptide described herein. The amount of glycopeptide present in a composition can vary. For instance, the dosage of glycopeptide can be between 0.01 micrograms (pg) and 3000 milligrams (mg), typically between 10 pg and 2000 pg. For an injectable composition (e.g. subcutaneous, intramuscular, etc.) the glycopeptide can be present in the composition in an amount such that the total volume of the composition administered is 0.5 ml to 5.0 ml, typically 1.0-3.0 ml. The compositions can be administered one or more times per day to one or more times per week, including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the condition, previous treatments, physical condition, health, age, weight, type and extent of the disease or disorder of the recipient, frequency of treatment, the nature of concurrent therapy, if required, and the nature and scope of the desired effect(s). Moreover, treatment of a subject with an effective amount of an active compound can include a single treatment or, preferably, can include a series of treatments. Such factors can be determined by one skilled in the art.

A composition including a pharmaceutically acceptable carrier can also include an adjuvant. An “adjuvant” refers to an agent that can act in a nonspecific manner to enhance an immune response to a particular antigen, thus potentially reducing the quantity of antigen necessary in any given immunizing composition, and/or the frequency of injection necessary in order to generate an adequate immune response to the antigen of interest. Adjuvants may include, for example, Freund’s (complete or incomplete), IL-1, IL-2, emulsifiers, muramyl dipeptides, dimethyldiocradecylammonium bromide (DDA), avridine, aluminum hydroxide, oils, saponins, alpha-tocopherol, polysaccharides, emulsified paraffins (available from under the tradename EMULSIGEN from MVP Laboratories, Ralston, Nebraska), ISA-70, RIBI and other substances known in the art.

Additional agents can also be incorporated into a composition. In one embodiment, a composition can include a biological response modifier, such as, for example, IL-2, IL-4 and/or IL-6, TNF, IFN-alpha, IFN-gamma, and other cytokines that effect immune cells. In another embodiment, a composition can include an inhibitor of degradation of the glycopeptide.

A pharmaceutical composition can be included in a container, pack, or dispenser together with instructions for administration. In one aspect, a pharmaceutical composition can be included as a part of a kit.

Methods

Also provided are methods. In one embodiment, a method is for making glycopeptide described herein. In one embodiment, the method includes incubating a genetically modified cell under suitable conditions for expression of a glycopeptide. The cell can be, but is not limited to, a genetically modified cell that includes an exogenous coding region encoding the protein component of the glycopeptide and includes the cellular machinery to add the carbohydrate component and optionally process the carbohydrate component. In one embodiment, a genetically modified cell can be used to produce a glycopeptide monmer. In one embodiment, a genetically modified cell can be used to make a multimer that is then cleaved into shorter glycopeptides. Kifunensine, a mannosidase inhibitor of the ER and Golgi mannosidase I, can be added during expression in 293 -F cells to produce glycopeptides with high mannose or oligomannose structures. Optionally, the method includes introducing into a host cell a vector that includes a coding region encoding the protein component. In one embodiment, the method includes isolating or purifying the glycopeptide from a cell or from a medium in which the cell is incubated. In those embodiments where the glycopeptide includes additional amino acids useful for isolating or purifying the protein, the method can also include cleavage of the additional amino acids from the glycopeptide.

In one embodiment, a method includes administering to a subject an effective amount of a composition including a glycopeptide described herein. As used herein, an “effective amount” of a composition including a glycopeptide described herein is the amount able to elicit the desired response in the recipient. The subject can be, for instance, murine (e.g., a mouse or rat), or a primate, such as a human.

In one embodiment, a method includes inducing an immune response to the glycopeptide by administering to a subject an effective amount of a composition including a glycopeptide described herein. In this embodiment, an “effective amount” is an amount effective to result in the production of an immune response in the subject. The immune response can be humoral, cell-based, or a combination thereof. A humoral immune response includes the production of antibodies that are antigen-specific and bind the glycopeptide used to induce the immune response. Methods for determining whether a subject has produced antibodies that specifically bind a glycopeptide described herein can be determined using routine methods. A cell-based response includes the production of T cells that produce interleukin-4 and/or interferon-gamma after stimulation by the glycopeptide used to induce the immune response.

As used herein, an antibody that can “specifically bind” a protein is an antibody that interacts with the epitope of the glycopeptide that induced the synthesis of the antibody or interacts with a structurally related epitope. It is expected that some of the epitopes present a glycopeptide described herein are epitopes that are conserved in glycopeptides of different clades of HIV.

In one embodiment, a method includes treating an infection in a subject caused by HIV. As used herein, the term “infection” refers to the presence of HIV in a subject’s body, which may or may not be clinically apparent. Treating an infection can be prophylactic or, alternatively, can be initiated after the subject is infected by the virus. Treatment that is prophylactic — e.g., initiated before a subject is infected by the virus or while any infection remains subclinical — is referred to herein as treatment of a subject that is “at risk” of infection. As used herein, the term “at risk” refers to a subject that may or may not actually be infected by the virus. Thus, typically, a subject “at risk” of infection by a virus is a subject that is a member of a population at increased risk of being exposed to the virus. Accordingly, administration of a composition can be performed before, during, or after the subject has first contact with the virus. Treatment initiated after the subject’s first contact with the virus may result in decreasing the severity of symptoms and/or clinical signs of infection by the virus, completely removing the virus, and/or decreasing the likelihood of experiencing a clinically evident infection. As used herein, the term “symptom” refers to subjective evidence of a disease or condition experienced by a subject and caused by infection by HIV. As used herein, the term “clinical sign” or, simply, “sign” refers to objective evidence of disease or condition caused by infection by HIV. Symptoms and/or clinical signs associated with conditions referred to herein and the evaluations of such symptoms are routine and known in the art.

The method includes administering an effective amount of a composition described herein to a subject having, or at risk of having, an HIV infection. In one embodiment, whether the viral load has decreased is determined. In this embodiment, an “effective amount” is an amount effective to reduce the viral load in a subject, or reduce the likelihood that the subject experiences a clinically-evident infection. Methods for determining whether an infection is caused by HIV are routine and known in the art, as are methods for determining whether the infection has decreased.

In another embodiment, a method includes treating one or more symptoms or clinical signs of certain conditions in a subject that may be caused by infection by HIV. The method includes administering an effective amount of a composition described herein to a subject having or at risk of having a condition, or exhibiting symptoms and/or clinical signs of a condition, and determining whether at least one symptom and/or clinical sign of the condition is changed, preferably, reduced.

A method of the present disclosure can further include additional administrations (e.g., one or more booster administrations) of the composition to the subject to enhance or stimulate a secondary immune response. A booster can be administered at a time after the first administration, for instance, one to eight weeks, such as two to four weeks, after the first administration of the composition. Subsequent boosters can be administered one, two, three, four, or more times annually.

Optionally, one or more other antigen, referred to here as a “secondary' antigen,” can be administered. Examples of suitable secondary antigens include, but are not limited to, other proteins and/or glycopeptides that are produced during an HIV infection, and proteins and/or glycopeptides that include epitopes present in the glycopeptide administered to result in an immune response. Examples include, but are not limited to, a protein and/or glycopeptide based on an HIV gpl40, such as a trimer envelope protein. In one embodiment, the secondary antigen is administered at the same time as a glycopeptide described herein, in the same composition or separately. In another embodiment, the secondary antigen is administered after the glycopeptide is administered. For instance, the secondary antigen can be administered before, at the same time as, or after a booster of the glycopeptide is administered.

HIV targets the CD4+ population of T cells in a subject, and infection typically results in the subject being unable to mount a cell mediated immune response to foreign antigens. In one embodiment, a glycopeptide of the present disclosure is administered after the subject has received therapy to increase the CD4+ population of T cells. Therapeutic agents and regimes that can be used to increase the CD4+ population of T cells in a subject infected with HIV are known to the skilled person.

Kits

Also provided are kits. In one embodiment, a kit is for using a glycopeptide described herein, such as using a glycopeptide to induce an immune response, or treat an infection, condition, symptom or sign. In one embodiment, a kit is for making a glycopeptide described herein.

The kit includes at least one of the glycopeptides described herein (e.g., one, at least two, at least three, etc.) or a multimer of at least one of the glycopeptides, or a genetically engineered cell that can express a glycopeptide described herein in a suitable packaging material in an amount sufficient for at least one assay or use. Optionally, other reagents such as buffers and solutions are also included. Instructions for use of the packaged glycopepide or cell are also typically included. As used herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit. The packaging material is constructed by routine methods, generally to provide a sterile, contaminant-free environment. The packaging material may have a label which indicates how a glycopeptide described herein can be used. In addition, the packaging material can contain instructions indicating how the materials within the kit are employed to administer a glycopeptide to an animal. As used herein, the term “package” refers to a container such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits the proteins, and other reagents, for instance a secondary antibody. “Instructions for use” typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like.

EXEMPLARY EMBODIMENTS

Embodiment 1. An isolated glycopeptide comprising an amino acid sequence having structural similarity to the amino acid sequence LDVVPIDNNNTSY (SEQ ID NO:l), wherein the residue at position 10 of SEQ ID NO:l comprises a glycan.

Embodiment 2. An isolated glycopeptide comprising an amino acid sequence having structural similarity to an amino acid sequence of at least 7 consecutive amino acids of a human HIV-gpl20 protein.

Embodiment 3. The isolated glycopeptide of embodiment 1 or 2 wherein the glycopeptide comprises no greater than 20 amino acids.

Embodiment 4. The glycopeptide of any of embodiments 1-3 wherein the glycan comprises a mannose.

Embodiment 5. The glycopeptide of any of embodiments 1-4 wherein the glycan comprises 2, 3, 4 or 5 mannose residues. Embodiment 6. The gly copeptide of any of embodiments 1-5 wherein the gly copeptide reacts with mannose-specific lectin Concanavalin A.

Embodiment 7. The gly copeptide of any of embodiments 1-6 wherein the gly can does not comprise one or more of a fucose saccharide or a sialic acid.

Embodiment 8. The glycopeptide of any of embodiments 1-7 wherein the glycan is an N- linked glycosylation.

Embodiment 9. The glycopeptide of any of embodiments 1-8 wherein the glycopeptide is a multimer.

Embodiment 10. The glycopeptide of any of embodiments 1-9 wherein the glycopeptide is a fusion protein comprising a heterologous amino acid sequence.

Embodiment 11. The glycopeptide of any of embodiments 1-10 wherein the heterologous amino acid sequence comprises a linker sequence.

Embodiment 12. The glycopeptide of any of embodiments 1-11 wherein the heterologous amino acid sequence comprises a cleavable sequence.

Embodiment 13. The glycopeptide of any of embodiments 1-12 wherein the cleavable sequence comprises an acid-labile sequence or a protease recognition sequence.

Embodiment 14. The glycopeptide of any of embodiments 1-13, the glycopeptide further comprising a lysine at the amino-terminal end of SEQ ID NO: 1.

Embodiment 15. The glycopeptide of any of embodiments 1-14, the glycopeptide further comprising an arginine at the amino-terminal end of SEQ ID NO: 1. Embodiment 16. A composition comprising the glycopeptide any of embodiments 1-15.

Embodiment 17. The composition of embodiment 16 further comprising a pharmaceutically acceptable carrier.

Embodiment 18. The composition of any of embodiments 16-17 further comprising an adjuvant.

Embodiment 19. A genetically engineered cell comprising an exogenous polynucleotide comprising a coding region encoding the protein component of the glycopeptide of any of embodiments 1-18.

Embodiment 20. The genetically engineered cell of embodiment 19 wherein the coding region is expressed and the protein is processed to comprise a glycan.

Embodiment 21. The cell of any of embodiments 19-20 wherein the cell comprises a N- acetylglucosaminyltransferase I mutation (GnTI-/-).

Embodiment 22. The cell of any of embodiments 19-21 wherein the cell stably expresses the glycopeptide.

Embodiment 23. A method comprising incubating the genetically engineered cell of any of embodiments 19-22 under conditions suitable for expression of the glycopeptide.

Embodiment 24. The method of embodiment 23 further comprising isolating the glycopeptide.

Embodiment 25. A method comprising: administering to a subject an amount of the composition of any of embodiments 16-18 effective to induce in the subject an immune response to the glycopeptide. Embodiment 26. The method of embodiment 25 wherein the immune response comprises the production of antibody that specifically binds to the glycopeptide.

Embodiment 27. The method of any of embodiments 25-26 wherein the immune response comprises the production of T cells that produce interleukin-4, interferon-gamma, or a combination thereof, after stimulation by the glycopeptide.

Embodiment 28. The method of any of embodiments 25-27 wherein the subject has or is at risk of having an infection caused by HIV.

Embodiment 29. A method for treating an infection in a subject, the method comprising: administering an effective amount of the composition of any of embodiments 16-18 to a subject having or at risk of having an infection caused by HIV.

Embodiment 30. A method for treating a symptom or a sign in a subject, the method comprising: administering an effective amount of the composition of any of embodiments 16-18 to a subject having or at risk of having an infection caused by HIV.

Embodiment 31. A method for treating a condition in a subject, the method comprising: administering an effective amount of the composition of any of embodiments 16-18 to a subject having or at risk of having a condition caused by HIV.

Embodiment 32. The method of any one of embodiments any of embodiments 25-31 wherein the HIV is a member of clade A.

Embodiment 33. The method of any one of embodiments any of embodiments 25-31 wherein the HIV is a member of clade B.

Embodiment 34. The method of any one of embodiments 25-33 further comprising a booster administration. Embodiment 35. The method of embodiment 34 wherein the booster comprises an envelope trimer.

Examples

The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

Example 1

Gly copeptide epitope facilitates HIV-1 envelope specific humoral immune responses by eliciting T cell help

The inherent molecular complexity of human pathogens requires that mammals evolved an adaptive immune system equipped to handle presentation of nonconventional MHC ligands derived from disease-causing agents, such as HIV envelope (Env) glycoprotein. Here, we report that a CD4+ T cell repertoire recognizes a gly copeptide epitope on gpl20 presented by MHCII pathway. This glycopeptide is strongly immunogenic in eliciting glycan-specific cellular and humoral immune responses. The glycopeptide specific CD4+ T cells display a prominent feature of Th2 and Thl7 differentiation and exert high efficacy and potency to help Env trimer humoral immune responses. Glycopeptide-induced CD4+ T cell response prior to Env trimer immunization elicits neutralizing antibody development and production of antibodies facilitating uptake of immunogens by antigen-presenting cells (APCs). The identification of gpl20 glycopeptide-induced, T cell-specific immune responses offers a foundation for developing future knowledge-based vaccines that elicit strong and long-lasting protective immune responses against HIV infection. This Example is also available as Sun et ak, 2020, Nature Communications, 11:2550 (doi: 10.1038/s41467-020- 16319-0). Introduction

Acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus- 1 (HIV-1) remains the most common cause of death from an infectious agent (1). The functional envelope spike protein of HIV-1 is a trimer of heterodimers composed of gpl20 proteins, each non-covalently associated with a transmembrane gp41 protein and is the primary target for host immune recognition (2-4). HIV Env trimer is highly glycosylated with glycans contributing to nearly half of its molecular weight (5). An increasing number of studies have illustrated that in addition to providing a shield to avoid immune responses, gpl20 glycans are the major “sites of vulnerability” targeted by broadly neutralizing antibodies (bNAbs) (6-8), which are the most effective and promising solution for protection against infection and suppression of established HIV-1 infection. In addition, several non-human primate (NHP) studies (9-11) and the RV144 human clinical HIV vaccine trial that exhibited a moderate protective effect against HIV acquisition (12-14) extend our understanding on the correlates of protection against viral challenge conferred by vaccine-induced functional non-neutralizing antibody responses.

Past and current research has so far gained in-depth insight on structural aspects ofB-cell receptor/antibody recognition of gpl20 epitopes (2,3,6,7,15). Major efforts to elicit protective antibody production have been devoted to sophisticated immunogen design (16), especially the development of recombinant native-like Env trimers (17-20). However, current HIV research does not leverage advances in knowledge related to maximizing stimulation of helper T cells to induce T cell-dependent humoral immune responses to the viral envelope. Despite broad appreciation that glycosylation of HIV gpl20 influences the repertoire of antibody responses elicited in infected individuals and that the epitopes recognized by many broadly neutralizing antibodies are glycan dependent, the importance of glycopeptides as non-conventional MHC ligands for generating T cell-mediated immunity to HIV has not been addressed (6-8). To illuminate the mechanisms of T cell-mediated immunity to the HIV envelope, we have explored the interactions of gpl20 glycopeptides with the adaptive arm of the immune system. Our previous work has characterized the molecular mechanisms by which glycoconjugate vaccines induce glycan-specific adaptive immune responses (21-23). Here, we report the existence of a CD4+ T cell repertoire that specifically recognizes gpl20 glycopeptide epitopes (i.e., glycotopes). We have identified a gpl20 glycopeptide presented by MHCII pathway serving as CD4+ T cell epitope. This glycopeptide elicits a glycan-specific cellular and humoral immune response. Glycopeptide stimulation also modulates T helper (Th) cell differentiation programming. Functionally, these glycopeptide specific CD4+ T cells play an important role in helping the humoral immune response to the HIV envelope glycoprotein, which indicates that initiating potent CD4+ T cell responses by glycopeptide epitopes may be an important component of future HIV vaccine strategies.

Results

CD4+ T cells recognize glycopeptide-epitopes of gpl20.

We first determined whether there exists a population of CD4+ T cells that specifically recognize glycopeptide epitopes on gpl20. For generation of immunogens enriched for glycopeptide epitopes, digestion of gpl20 by endoproteinase Glu-C was followed by chromatographic separation and lectin selection (Fig. 1A). Immunization of BALB/c mice with pooled gpl20 glycopeptides induced gpl20-specific IgG production (Fig. 1A). CD4+ T cells were isolated from mice immunized with gpl20 glycopeptide pool and were stimulated in vitro with intact gpl20 or with deglycosylated gpl20 (DG-gpl20) — i.e., gpl20 treated with peptide N-glycosidase F (PNGase F) to remove N-glycans (Fig. IB) — in the presence of APCs. Total T cell proliferation was measured as the frequency of carboxyfluorescein diacetate succinimidyl ester (CFSE)low cells. CD4+ T cells stimulatwith intact gpl20 had a significantly higher proliferation rate, as evidenced by a higher percentage of CFSElow cells in the CD4+ T cell population, than T cells stimulated with DGgpl20 (Fig. 1A, B); this difference provided evidence for a glycopeptide-specific CD4+ T cell proliferative response. Intact glycosylated gpl20 inhibited binding of antisera from mice immunized with the gpl20 glycopeptide pool, whereas DG-gpl20 did not inhibit antiserum binding to intact gpl20 in a competition ELISA (Fig. 1C). To further characterize the gpl20 glycopeptide-stimulated T cell population, we collected supernatant from each stimulation group for quantification of interleukin 4 (IL-4) and interferon g (IFN-g) production. While CD4+ T cells stimulated with gpl20 produced more Th2- associated cytokine IL-4, those stimulated with DG-gpl20 produced more Thl -associated IFN-g (Fig. ID, E). We assessed the serum IgG subclasses associated with gpl20 and DG-gpl20 specificity. In line with the cytokine profiles, the gpl20-specific IgG response consisted predominantly of the IgGl subclass, while DG-gp 120-binding IgG was predominantly of the IgG2a and IgG3 subclasses (Fig. 1F-H). Taken together, these data demonstrate that certain populations of CD4+ T cells differentiate between glycosylated and non-glycosylated gpl20 epitopes, indicating carbohydrate recognition by CD4+ T cells. Moreover, gpl20 glycopeptide stimulation can modulate Th cell differentiation programming.

Identification of gpl20 glycopeptide epitopes presented by MHCII pathway.

To determine whether gpl20 gly cotopes are presented by MHCII, we immunoprecipitated MHCII from APCs (mouse bone marrow-derived dendritic cells (BMDCs)) and identified bound epitopes by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). BMDCs were co-incubated with glycosylated gpl20 expressed in 293-F cells. After cell lysis, MHCII proteins were immunoprecipitated with a monoclonal antibody specific for mouse MHCII molecules I-A and I-E. MHCII-bound epitopes were released and treated with PNGase F in the presence of 180-H20, which marks glycosylated asparagine residues by their conversion to aspartate and incorporation of a heavy oxygen atom. Epitopes released from MHCII were analyzed directly by LC-MS/MS or were digested with one of two proteases

(try psin or chymotrypsin) to enhance sequence coverage and peptide detectability. A variety of glycopeptides and peptides were identified (Table 1). A glycopeptide epitope derived from the second variable region (V2) of gpl20 — KLDVVPIDNNN187TSY, glycosylated atN187 and designated GpepIP — was identified in protease-treated and untreated pools of released epitopes, exhibiting single amino-acid extensions or truncations at the N- or C-terminus (Fig. 3A-C, Table 1). As revealed by our previous comprehensive glycomic and glycoproteomic characterization of the gpl20 used in this study (24), the predominant glycan structure at the N187 site is Man5GlcNAc2 (M5N2, Fig. 4) along with other minor oligomannose, hybrid, and complex glycoforms. To obtain the dominant glycoform of this epitope, we recombinantly expressed polyhistidine (His)-tagged GpepIP in N-acetylglucosaminyltransferase I mutant (GnTI-/-) cells, which produce exclusively oligomannose N-glycans (25). In addition, we generated a GpepIP variant with predominantly complex glycans on N187 by recombinant expression in 293-F cells. The non-glycosylated peptide backbone (pepIP) was synthesized by solid-state peptide synthesis. All three GpepIP variants were separated by SDS-PAGE, and glycosylation of GpepIP was verified by the molecular- weight shift after PNGase F treatment (Fig. 5 A, B). GpepIP — but not pepIP — reacted with the mannose-specific lectin concanavalin A (ConA, Fig. 5C). The glycan structures on recombinant GpepIP prepared in GnTI-/- cells were confirmed by LC-MS/MS (Fig. 6A, B). The major N-glycan on GpepIP was M5N2, with M4N2, M4N2F, and M5N2F as minor components (Fig. 6B). GpepIP from 293-F cells expressed almost exclusively complex-type glycans with extensive branching, fucosylation, and sialylation (Fig. 7).

Table 1. MHCII-bound gpl20 glycopeptides/peptides To further explore the structural specificity of the N187 glycan’ s impact on GpepIP binding to MHCII, GpepIP variants were exchanged onto recombinant mouse MHCII I- Ad molecules immediately after protease cleavage of CLIP placeholder peptide (26). MHCII molecules with empty binding grooves aggregate during prolonged incubation (27); thus, epitope-MHCII binding can be assessed by western blotting for intact MHCII dimer after resolution by isoelectric focusing (IEF) gel electrophoresis (Fig. 3D). OVA peptide 323-339, a well-characterized MHCII-binding epitope, was used as a positive control, while its scrambled version was used as a negative control for MHCII binding. GpepIP expressed in GnTI-/- cells bound to MHCII with or without a His tag at its N-terminus (Fig. 3D). This observation agrees with the fact that MHCII, structurally different from MHCI, can accommodate larger peptides (i.e., >10 amino acids), as the binding groove is open and allows for greater flexibility (28).

PepIP with a His tag also bound to MHCII as predicted. However, GpepIP expressed in 293-F cells, modified predominantly with complex glycans, did not bind measurably to MHCII. To confirm the presence of MHCII-epitope complexes, we excised bands corresponding to GpepIP/MHCII, pepIP/MHCII, and OVA peptide/MHCII complexes from IEF gels and assayed for peptide and glycopeptide epitopes by LC-MS/MS (Fig. 3E). MHCII a and b chain peptides were also detected (data not shown).

GpepIP elicits glycan-specific cellular and humoral immune response.

In an assessment of whether GpepIP binding to MHCII elicits cellular and humoral adaptive immune responses, CD4+ T cells isolated from gpl20-immunized mice were stimulated with GpepIP or pepIP. The greater T cell proliferation in response to GpepIP than in response to pepIP indicated that GpepIP is a gpl20 epitope recognized by CD4+ T cells in a glycan- dependent manner (Fig. 8A, B). CD4+ T cells isolated from mice immunized with GnTI-/— expressed GpepIP were predominantly stimulated by GnTI-/- expressed GpepIP rather than by pepIP (Fig. 8C). This result strongly supports glycan participation in the CD4+ T cell response to the GpepIP epitope; in addition, 293-F expressed GpepIP did not stimulate T cell responses (Fig. 8C), possibly because of poor MHCII binding (Fig. 8C). Alternatively, the enhanced potency detected for GnTI-/-expressed GpepIP over that of 293-F cells can be due to T cell receptor recognition of specific oligomannosidic glycan motifs. When mice were immunized with pepIP, their CD4+ T cells responded only to peptide without glycans; adding glycans of any type to this epitope (GpepIP expressed in GnTI-/- or 293-F cells) blocked pepIP-specific CD4+ T cell responses (Fig. 8D). These results revealed that recognition of the GpepIP epitope by CD4+ T cells is glycan dependent.

We next evaluated T cell-dependent antibody responses induced by GpepIP and pepIP immunization in terms of binding to glycosylated and non-glycosylated epitopes. Antisera from GpepIP -immunized mice exhibited remarkably strong binding to GpepIP (Fig. 8E), whereas pepIP immunization predominantly produced high-affinity antibodies to pepIP (Fig. 8F). Importantly, antisera from GpepIP-immunized mice showed significantly greater binding to intact gpl20 expressed in either 293-F or GnTI-/- cells than pepIP antisera (Fig. 8G). Lack of a differential ELISA response for GnTI-/- gpl20 and even for 293-F gpl20 is likely due to oligo/high-mannose structures displayed on gpl20, including the GpepIP site (Fig. 4). We also assessed the IgG subclasses of GpepIP -immunized antisera associated with gpl20 and DG- gpl20 specificity. GpepIP immunization induced higher IgGl and IgG2a subclasses of gpl20- specific IgG than DG-gp 120-specific IgG subclass (Fig. 8H, I). gpl20- and DG-gpl20-specific IgG3 subclass appeared comparable (Fig. 81). Taken together, these results demonstrate that the GpepIP epitope induces glycopeptide-specific CD4+ T cell and antibody responses, and it also modulates CD4+ T cell differentiation. GpepIP induces preferentially Th2 and Thl7 differentiation.

To further characterize the CD4+T cell responses induced by the non-conventional MHC ligand — GpepIP — and to obtain a gene expression profile of GpepIP-induced CD4+ T cells, we performed genome-wide transcriptomic analysis (RNA-seq) comparing GpepIP and pepIP - induced CD4+ T cell responses. Splenocytes from GnTI-/-expressed GpepIP and pepIP immunized mice were stimulated in vitro with GpepIP and pepIP respectively for three days. Using T cell activation marker CD69, we sorted out CD4+CD69+ T cell populations by flow cytometry whereas CD4+CD69- non-responding cells were used as control (Fig. 9A). High cd4 gene expression was observed in all sorted groups and cd69 and 112 were upregulated in GpepIP - and pepIP-stimulated groups compared to control (data no shown). Hierarchical clustering of genes from each group revealed three distinct gene expression patterns with closer similarities between GpepIP and pepIP cells than with control (Fig. 9A). Comparing transcriptomes of GpepIP and control cells, we found that 3001 genes were differentially expressed (greater than twofold, P < 0.05) with 2460 genes upregulated, 541 genes downregulated and 12711 genes unchanged (Fig. 9C left and data not shown). Gene Ontology (GO) analysis showed a large fraction of differentially expressed genes (DEGs) in GpepIP group enriched in the biological processes associated with cellular process, metabolic process, response to stimulus, signaling and immune system process (Fig. 10A). Kyoto encyclopedia of genes and genomes (KEGG) pathways analysis of DEGs identified the enriched pathways were highly associated with immune functions, such as pathways in cancer, inflammatory bowel disease (IBD), hematopoietic cell lineage, cytokine-cytokine receptor interaction and leishmaniasis.

Particularly, GpepIP specific CD4+ T cells were enriched for Thl, Th2 and Thl7 cell differentiation compared to control (Fig. 9D left). Detailed examination of the top hits revealed a significant enrichment for genes associated with Thl and Th2 signaling (Ifng, 112, Il2ra, Ilia, Illr2, 114, 116, 119, 1110, 1113, Mcptl), Thl7 signaling (1117a, I117f, 1122, 1123r, Iltifb, D114, Cxcl3) and activated T cell costimulatory signaling (cd44, cd63, cd83, Icos) (Fig. 10B).

Comparing transcriptomes of pepIP to control T cells, 2620 genes were differentially expressed with 1950 genes upregulated and 670 genes downregulated (Fig. 9C middle and data not shown). KEGG pathways analysis of DEGs of pepIP-specific CD4+ T cells also exhibited an enrichment of Thl, Th2 and Thl7 cell differentiation (Fig. 9 D middle). However, when comparing transcriptomes between GpepIP and pepIP specific CD4+ T cells, only 620 genes were differentially expressed (615 up-regulated and 205 downregulated) (Fig. 9C right and data not shown). Notably, IL-17 signaling pathway was favorably enriched in GpepIP induced CD4+ T cells (Fig. 9D right). The transcription signatures associated with Thl and Th2 cell differentiation, IL-17 signaling pathway and Thl 7 cell differentiation as noted in KEGG analysis were summarized as a heatmap normalizing to control (Fig. 9E). Prominent genes associated with Thl differentiation appeared to have comparable expression levels in GpepIP and pepIP- specific CD4+ T cells, including Ifng, 112, II 18rap, Setbpl, Nkg7, cd86, Ccl4, 1112, Statl and Tbx21 (encoding T-bet) (Fig. 9E). Prominent genes associated with Th2 differentiation, however, were highly upregulated in GpepIP compared to pepIP induced CD4+ T cells, such as 115, 116, 119, 1110, 1113, Nlrp3, Illrll, Cypl lal, Mcptl and Serpinb2 (Fig. 9E, F). Of note, produced by both Th2 and follicular helper T (Tfh) cells 29, the expression of 114 showed no difference between GpepIP and pepIP (Fig. 9E). Strikingly, the expression of genes associated with Thl7 signature was remarkably elevated in GpepIP-specific CD4+ T cells, including 1117a, I117f, 1122, Iltifb, I123r, Rorc (encoding RORyt), Cxcll, Cxcl3, Lcn2, D114, Illrl, Saa3, Nos2, Ptges, Lum and Idol (Fig. 9E, F), indicating a robust Thl 7 differentiation elicited by GpepIP.

The Th cell differentiation status of GpepIP and pepIP specific CD4+ T cells was further validated at the protein level by assessing Thl, Th2 and Thl7 signature cytokines in T cell cultured supernatant. After a 5-day GpepIP or pepIP antigen stimulation of T cells from GpepIP or pepIP immunized mice, supernatants were harvested for a multiplex-based cytokine measurement. Consistent with RNA-seq data, both GpepIP and pepIP stimulated supernatants displayed significantly increased Thl and Th2 cytokines production compared to medium group (Fig. 11 A, B). Despite the Th2 enrichment in both GpepIP and pepIP groups, signature cytokines after GpepIP stimulation showed markedly augmented expression, such as IL-5, IL-6, IL-10 and IL-13 (Fig. 11C). Yet, similar IL-4 expression was observed in both groups (Fig. 11C). Although pepIP stimulation induced increased IL-17A production over medium alone, the extent of its expression was strikingly lower than GpepIP groups (Fig. 11C). Additionally, the expression levels of two other Thl7-related cytokines IL-17F and IL-22 were substantially lower in pepIP than GpepIP group (Fig. 11C). In addition, representative Thl, Th2 and Thl7 cytokines IFN-g, IL-5 and IL-17A were evaluated by intracellular cytokine staining combined with flow cytometry. The results showed that after three days of antigen stimulation, GpepIP specific CD4+ T cells displayed an overall higher expression of all three cytokines with greater enhancement in Th2 and Thl7 cytokines (Fig. 1 ID, E). The higher IFN-g production in GpepIP than pepIP specific CD4+ T cells detected at the protein level (Fig. 11) are not in accord with mRNA results (Fig. 9); this is likely at least partially due to different time points of sample collection. As the cytokines were detected on day 5 after antigen stimulation, IFN-g production was comparable between GpepIP and pepIP groups, albeit IL-5 and IL-17A were still higher in GpepIP specific CD4+ T cells (Fig. 12A, B). Taken together, transcriptomic analysis and cytokine assessment results demonstrate that the pepIP epitope activates mainly Thl polarized CD4+ T cells whereas glycosylation on this epitope (GpepIP) induces a distinct and strong Th2 and Thl 7 enrichment.

GpepIP specific CD4+ T cells exert superior effects on helping HIV envelope specific humoral immune responses.

CD4+ T cells are essential mediators in helping B cells and antibody responses (29). We next sought to assess whether GpepIP-induced CD4+ T cell facilitates antibody responses to Env trimer — a more immunologically relevant challenge. The B cell-help by pepIP-specific CD4+ T cells was also investigated to determine the specificity and potency of glycopeptide T cell epitopes over peptide epitopes. BALB/c mice were primed with GnTI-/-expressed GpepIP or pepIP emulsified in Freund’s adjuvant or with adjuvant alone; all groups were boosted with a clade A BG505 gpl40 native flexibly linked (NFL) trimer (30). Antisera were collected at specific time points (Fig. 13A). GpepIP- and pepIP-specific CD4+ T cells were induced in GpepIP- and pepIP-primed mice respectively, compared to adjuvant group, as evidenced by significantly greater T cell proliferation in the GpepIP- and pepIP-primed groups after in vitro stimulation with GpepIP and pepIP (Fig. 13B). The stimulation of T cells by GpepIP in the adjuvant-primed group is likely due to the response to the Env trimer after BG505 booster immunization (Fig. 13B). With distinct antigen specific CD4+ T cell help, Env trimer-specific IgG production was compared in these mice across the indicated time points (Fig. 13C). Substantially higher BG505-specific IgG titers were observed in mice primed with GpepIP than with adjuvant or pepIP (P < 0.0001) (Fig. 13C). The help from pepIP priming was different from adjuvant priming only at the experimental endpoint (Fig. 13C). In agreement with serum IgG titers, Env trimer specific B-cell responses were enhanced in GpepIP -primed mice as well. Compared to adjuvant and pepIP, GpepIP primary immunization induced superior germinal center (GC) response, defined by a significantly increased percentage of GL7+Fas+ B cells (Fig. 13D). Moreover, the expression of B cell activation markers CD69, CD80 and MUCH was higher among splenic B cells isolated from GpepIP primed mice after in vitro stimulation with BG505 trimer (Fig. 13E-G). These results indicate that GpepIP -induced CD4+ T cells provide effective and potent help for Env trimer antibody responses compared to pepIP specific CD4+ T cells.

GpepIP specific CD4+ T cells stimulate enhanced functional antibody responses.

With the observation of GpepIP eliciting HIV envelope-specific humoral immune responses, we next sought to determine the contribution of GpepIP-induced CD4+ T cells to the functional antibody development. We applied an immunization regimen with three prime immunizations of adjuvant, GpepIP or pepIP, followed by three boost immunization of BG505 to improve GpepIP-induced CD4+ T cell help and Env trimer-specific IgG responses (Fig. 14A). Env trimer-specific booster antibody responses were assessed by ELISA (Fig. 14B). In agreement with previous results (Fig. 13C), GpepIP priming significantly increased BG505 IgG production than adjuvant or pepIP priming groups after first trimer boost (Post 1). The IgG titers of GpepIP- and pepIP -priming groups were similar after second trimer boost (Post 2), while both were significantly higher than adjuvant group. After third BG505 immunization, all three groups had comparable BG505 IgG titers (Post 3). We then evaluated the quality and function of antisera after three trimer immunizations of all tested groups. Different effector T helper subsets have been identified to promote antibodies class switching (31, 32). Antisera from all three groups after BG505 boost for three times — with equivalent BG505 IgG titers — were tested for their IgG subclasses. Among all, IgGl subclass represented the predominant antibody subclass in all immunization groups without significant difference between groups. While pepIP priming induced higher BG505 IgG2a subclass, GpepIP specific CD4+ T cells preferentially helped BG505 IgG class switching towards IgG2b and IgG3 (Fig. 14C).

The neutralizing activity of these sera was then assessed via a TZM-bl cell-based neutralization assays (33, 34). Antisera from all tested groups after three BG505 booster immunizations were assayed against tier 1 and tier 2 HIV-1 viruses. Compared to trimer immunization only (adjuvant), we detected clear neutralization of the tier 1 A virus MN.3 in all sera samples from GpepIP pre-immunization group (Fig. 14D). Notably, sera from pepIP pre-immunization did not also show neutralizing activities. Antisera from this pilot assay did not show broad neutralizing breadth. On the other hand, there is emerging evidence indicating that functional non-neutralizing antibodies provide effective protection against HIV infection (16). An antibody-mediated immunogen-uptake assay was established based on our previous method (24) to evaluate antibody function. With equivalent Env trimer specific IgG titers (Fig. 15), pre-incubation of fluorophore-labeled BG505 with antisera from GpepIP -primed and BG505-boosted mice significantly promoted the BG505 uptake by BMDCs compared to incubation with no serum, and more importantly, compared to sera from adjuvant- or pepIP-primed mice (Fig. 14E). Taken together, these data indicate that GpepIP epitope, but not the naked peptide epitope, elicits a repertoire of CD4+ T cells that help HIV envelope-specific-B cells to produce enhanced, protective and functional antibodies.

Discussion

Identification and characterization of broadly neutralizing antibodies (several of which recognize the glycan shield of gpl20 (2, 6-8, 35)) and of non-neutralizing polyfunctional antibodies (16, 36) from HIV-infected individuals have buoyed hopes that inducing the production of protective antibodies through immunization can halt the spread of infection.

Efforts toward intelligent implementation of this strategy have benefited from a deep understanding of B-cell receptor/envelope recognition (16, 37-41).

In this study, we addressed the other critical direction of HIV research: elucidation of gpl20-induced T cell activation mechanisms underlying elicitation of Env trimer-specific humoral immune responses. Recent studies have demonstrated critical roles for CD4+ helper T cells in driving antibody subclass switching, affinity maturation, and effector function of antibodies to HIV (29, 42, 43). Particularly, generation of bNAbs necessitate affinity maturation and somatic mutations, involving CD4+ T cell help 43. The RV144 vaccine trial showed a correlation of CD4+ T cell response with protection, indicating the contribution of helper T cells in protective antibody production (44).

Past efforts to identify gpl20 T cell epitopes focused solely on naked peptide epitopes (45). By using overlapping peptides spanning the entire gpl20, it was suggested that the promiscuously immunodominant gpl20 T cell epitopes were clustered forming CD4+ T cell epitope “hot spots” (46, 47). However, gpl20 proteins are heavily glycosylated with various numbers (from 23 to 26) of N-linked glycosylation sites across the protein region (48, 49). It has been shown that N-linked glycans on gpl20 influence CD4+ T cell responses by modulating antigen processing (50) or epitope generation (51). More importantly, within APCs, gpl20 glycan moieties survive the antigen processing, yielding glycopeptide epitopes presented by MHCII to CD4+ T cells. Therefore, gpl20 glycan shield will significantly skew CD4+ T cell epitope “hot spots”. Thus, it is plausible to evaluate the importance of gpl20 glycopeptidespecific CD4+ T cells. Additionally, the view that carbohydrates serve directly as nonconventional epitopes to induce CD4+ T cell responses have received increasing appreciation (21, 23, 26, 52). Here, we isolated and characterized a MHCII-presented glycopeptide epitope (GpepIP) eliciting T cell-mediated humoral immune responses to the HIV envelope glycoprotein. Our data demonstrate that gpl20 glycans directly participate in CD4+ T cell recognition, evidenced by: 1) loss of glycans impairs the T cell response to a glycopeptide epitope; 2) alteration of glycan structure dampens the T cell response; 3) addition of glycans blocks the T cell response to a peptide epitope.

Previous studies have reported that carbohydrate-specific signaling shapes T helper cell differentiation through modulating the crosstalk between APCs and CD4+ T cells (53-55). In this study, we identified Th cell differentiation programming upon glycopeptide epitope recognition. The comprehensive RNA-seq analysis and Th-associated cytokine profiles demonstrate that a peptide epitope (pepIP) induces a Thl dominant immune response, whereas recognition of a glycopeptide epitope (GpepIP) by CD4+ T cells drives the induction of Th differentiation towards Th2 and Thl7 features. Further investigation is needed to elucidate the mechanisms underlying regulation of CD4+ T cell differentiation and function by their glycan recognition.

Functionally, GpepIP epitope shows greater effectiveness and potency in eliciting T cell help for HIV envelope humoral immune response than pepIP epitope. Further, GpepIP (derived from clade B) induced CD4+ T cells display broader help breadth for a heterologous Env trimer BG505 (clade A). Tfh cells are CD4+ T cells specialized in helping B cell-mediated immunity and antibody responses (56). However, our data indicates that neither GpepIP nor pepIP specific CD4+ T cells are Tfh cells as the expression of genes associated with Tfh cell programing, such as Bcl6, Pdcdl (encoding PD-1), Sh2dla (encoding SLAM-associated protein (SAP)) and Btla showed no difference from control group; and minimal IL-21 production was detected. The superior antibody responses by GpepIP over pepIP is most likely due to GpepIP stimulating more effective Th2 and Thl7 responses than the pepIP (31, 57-59). GpepIP elicits substantial antibody response targeting gpl20 gly can-epitopes shared by immunogens across clades, further contributing to GpepIP specific CD4+ T cells’ potency. Analyses of RV144 vaccine trial identified a unique immune response profile, marked by V2-specific IgG3 antibodies and IL-13 signature from envelope stimulated PBMC supernatant (12), suggesting the functional potential of GpepIP elicited Th2 and IgG3 responses.

Importantly, as a proof-of-principle for driving functional antibody responses through eliciting glycopeptide-specific helper T cell activation, we demonstrated that GpepIP primary immunization followed by BG505 booster immunization resulted in tier 1 neutralizing antibody development, while BG505 booster immunization alone (adjuvant preimmunization) and pepIP pre-immunization did not. With equivalent IgG titers, BG505 antibodies from GpepIP -primed mice have a greater functional capability to mediate antigen uptake by APCs than adjuvant- or pepIP -primed mice. Thus, adaptive immune mechanisms induced by glycopeptides of the HIV envelope glycoprotein described here offer an innovative approach for future clinical development of knowledge-based, protective HIV vaccines. Materials and methods

Mice

Eight-week-old female BALB/c mice were obtained from Taconic Bio-sciences (Hudson, NY) and housed in the Coverdell Rodent Vivarium at the University of Georgia. Mice were kept in microisolator cages and handled under a laminar flow hood. All mouse experiments were conducted in compliance with the University of Georgia Institutional Animal Care and Use Committee under an approved animal use protocol.

Antigen production

The codon-optimized pSyn gpl20 plasmid encoding JR-FL (clade B) gpl20 was obtained from Dr. Eun-Chung Park and Dr. Brian Seed through the NIH AIDS Reagent Program, Division of AIDS, NTATD, NIH (60, 61). The HIV-1 BG505 gpl40 NFL expression vector was obtained from Dr. Richard T. Wyatt through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH (30). Both of them were expressed in a serum-free medium by transient transfection of wild-type FreeStyle™ 293-F suspension cells (Life Technologies); gpl20 and BG505 trimer were purified from supernatant by affinity chromatography using Galanthus nivalis lectin- agarose (Vector Labs, Burlingame, CA) and then further purified on a Superdex S200 size exclusion column (Bio-Rad) to isolate gpl20 and trimer fraction as described previously (24,

62). gpl20 was also expressed and purified from HEK293S GnTI-/- cells (ATCC).

To generate a gly copeptide pool, gpl20 was digested by sequencing-grade Endoproteinase Glu-C (Promega) in ammonium bicarbonate buffer. Digested products were separated on a Superdex Peptide 10/300 GL column (GE Healthcare) in order to selectively isolate glycopeptides from non-glycosylated peptides. Glycopeptide-containing fractions were identified and pooled together on the basis of biotinylated ConA (Vector Labs) reactivity in a lectin dot blot. Pooled glycopeptides were desalted on a HiPrep 26/10 Desalting column (GE Healthcare) and lyophilized overnight.

The gpl20 GpepIP expression vector was generated by cloning of the synthetic nucleotide sequence (GENEWIZ) that codes for the signal sequence-6xHistidine-GpepIP peptide into the pGEn2 restriction-site mammalian expression vector (provided by Dr. Kelley Moremen, University of Georgia) via Notl and BamHI restriction sites. GpepIP expression vector containing a His tag was generated. Recombinant GpepIP was expressed in transiently transfected 293-F cells or HEK293S GnTI-/- cells (ATCC) as described previously (24). GpepIP was purified by passage of supernatant through NΪ2+-NTA resin (G-bioscience) at 4°C, washing with buffer containing 10 mM imidazole (G-bioscience) and 0.2 M NaCl, and elution with 0.3 M imidazole. The elution was collected, desalted, and lyophilized for longterm storage. The purity of glycopeptides was evaluated by SDS-PAGE and silver staining (Pierce). Concentrations of GpepIP and pepIP were determined by the quantitative fluorometric peptide assay (Pierce) according to the manufacturer’s protocol. All peptides, including pepIP, OVA peptide 323-339, and scrambled OVA peptide, were purchased from GenScript (Piscataway, NJ).

For removal of N-linked glycans, PNGase F (a gift from Dr. Kelley Moremen, University of Georgia) (63) was applied under denaturing conditions as previously described (24). In brief, glycoproteins or glycopeptides were boiled in 20 mM sodium phosphate buffer (pH 7.5) containing 0.05% SDS and 50 mM b-mercaptoethanol. PNGase F was added to the protein at a ratio of 1 : 10; overnight incubation at 37°C followed.

Immunization

Groups of BALB/c mice were immunized on days 0 and 21 with 10 pg of gpl20 emulsified in Freund’s adjuvant (Thermo Scientific) by subcutaneous injection dorsal to the base of the tail. For glycopeptide or peptide immunization, 30 pg of antigen was used. During prime- boost immunization experiments, mice were bled from the tail vein 7 days after boosting. For evaluation of the role of GpepIP- and pepIP- specific CD4+ T cells in selectively eliciting trimer antibody production, mice were primed twice (with a 21 -day interval) with either GpepIP emulsified in Freund’s adjuvant or pepIP or adjuvant alone. 21 days after the second priming, both groups of mice were boosted with 20 pg of the BG505 trimer emulsified in incomplete Freund’s adjuvant. Sera were collected at the indicated time points.

For evaluation of functional antibody responses, BALB/c mice were primed three times (with a 3-week interval) by subcutaneous injection of GpepIP or pepIP emulsified in Freund’s adjuvant or of adjuvant alone. Three weeks later, all groups were boosted by intraperitoneal injection of 20ug of the clade A BG505 gpl40 NFL trimer adjuvanted with 2% alhydrogel (Invivogen) for three times (with a 3-week interval). Sera were collected 7 days after each trimer boost. T cell proliferation, B cell activation and flow cytometry

CD4+ T cells were isolated from lymph nodes of mice three weeks after booster immunization with a mouse CD4 T lymphocyte enrichment set (BD Biosciences) according to the manufacturer’s protocol. CD4+ T cells were stimulated in vitro in the presence of mitomycin C-treated (25 pg/ml, Sigma-Aldrich) splenic mononuclear cells pulsed with 10 pg/ml of indicated antigens (20 pg/ml for glycopeptide and peptide) for 5 days. In some experiments, total splenic and lymph node cells were used. For CFSE labeling, CD4+ T cells were incubated with 2 mM CFSE solution (Sigma-Aldrich) at 37 °C for 8 minutes before stimulation. CFSE dilution was measured by flow cytometry as an indication of the T cell proliferation rate.

To evaluate CD4+ T cell mediated B cell responses, mice were sacrificed 10 days after trimer booster imunization. GC response was assessed by the expression of GL7 and Fas among splenic B cells by flow cytometry. Splenocytes were stimulated in vitro with lOug/ml of BG505 trimer for 3 days to examine the expression of activation markers CD69, CD80 and MHCII by flow cytometry.

Surface staining of cell suspensions was performed by incubation of indicated mAbs for 30 min in PBS containing 0.1% BSA and 0.02% NaN3 at 4 ^° C in the dark (64). The following fluorophore-conjugated antibodies used in flow cytometry detection were purchased from BioLegend: anti-mouse CD4 (clone GK1.5), anti-mouse/human GL7 (clone GL7), antimouse CD95 (Fas) (clone SA367H8), anti-mouse CD69 (clone HI.2F3), anti-mouse CD80 (clone 16- 10A1), and anti-mouse I-A/I-E (clone M5/114.15.2). Fluorescent signal was analyzed on CytoFLEX (Beckman Coulter, Hialeah, FL). Data were analyzed with FlowJo software (Tree Star, Inc., Ashland, OR).

Cytokine analysis

After a 5-day GpepIP or pepIP antigen stimulation of T cells from GpepIP or pepIP immunized mice, supernatants were harvested, and the concentrations of Th cell associated cytokines were assessed using LEGENDplex mouse T helper cytokine immunoassay kit (BioLegend) according to the manufacturer’s instructions.

For intracellular cytokine staining, splenic and lymph node cells were isolated from GpepIP and pepIP immunized mice and stimulated in vitro with GpepIP and pepIP respectively for three or five days. GolgiPlug (BD Biosciences) was added at the recommended concentration in the last 6 hours of stimulation. Cells were collected and stained for surface marker CD4 (Biolegend). Intracellular staining of IFN-g, IL-5 and IL-17A was performed following fixation and permeabilization of stimulated cells with Cytofix/Cytoperm solution (BD Pharmingen) using the following antibodies: anti-mouse IFN-g (clone XMG1.2), anti-mouse/human IL-5 (clone TRFK5) and anti-mouse IL-17A (clone TCI 1-18H10.1). All antibodies and isotype fluorescence conjugated antibodies were purchased from Biolegend. Samples were analyzed on CytoFLEX (Beckman Coulter, Hialeah, FL). Data were analyzed with FlowJo software (Tree Star, Inc., Ashland, OR).

Enzyme-linked immunosorbent assay (ELISA).

Cytokine production resulting from T cell stimulation was measured by ELISA. In brief, 96-well plates (Costar) were coated overnight with antibody to IFN-g or IL-4 (1 : 1000 dilution; Biolegend) and blocked with 1% BSA/PBS. Plates were washed with 0.05% PBS-Tween and incubated with cell supernatants for 2 hours at room temperature. After washing, biotinylated detection antibodies to IFN-g or IL-4 (1 : 1000 dilution; Biolegend) were added for 2 hours at room temperature, after which HRP-conjugated Avidin (1 : 1000 dilution; Biolegend) was added for 1 hour at room temperature. Plates were developed with a 3,3',5,5'-tetramethylbenzidine substrate (TMB; Biolegend), and development was stopped with 2 N H2S04. The optical densities were determined at 450 nm with a microplate reader (Synergy HI, Bio-Tek).

Antigen-specific antibodies in sera were measured by ELISA. Briefly, 96-well plate was coated with anti-gpl20 mAb D7324 (Aalto Bioreagents, Dublin, Ireland) to capture gpl20 and BG505 trimer. GpepIP and pepIP (5 pg/ml) were directly coated onto 96-well plates. After washing and blocking steps, serial dilutions of serum from immunized mice were added to the plate for 2 hours at room temperature. Total IgG was detected by alkaline phosphatase (AP)- conjugated anti-mouse IgG (Southern Biotech) coupled with AP substrate (Sigma-Aldrich). The optical densities were determined at 405 nm. IgG subclasses were detected by HRP-conjugated anti-mouse IgGl, IgG2a, IgG2b and IgG3 (Abeam) and TMB substrate as described above.

To clarify epitope recognition of antisera from immunized mice, inhibition ELISA was performed. Sera diluted 1 :800 were pre-incubated with different concentrations of inhibitors before being added to a plate pre-coated with gpl20 protein (5 pg/rnl). IgG production was measured as described above.

Purification of MHCII-bound gly copeptides by immunoprecipitation

BMDCs were generated after GM-CSF (PeproTech) induction for 8 days as described previously (24) and were incubated with gpl20 protein (250 pg/ml) at 37°C for 18 hours. Cell lysate was prepared with the following lysis buffer (pH 8.0): 20 mM Tris-HCl, 137 mM NaCl, 1% Nonidet P-40 (NP-40), and 2mM EDTA with a protease inhibitor cocktail (AEBSF, Aprotinin, Bestatin, E-64, EDTA, Leupeptin) (Sigma-Aldrich). The protein concentration was measured by bicinchoninic acid analysis (Pierce).

Lysate was pre-cleared by incubation with isotype antibody and bead slurry at 4°C for 1 hour. Affinity purification of MHCII molecules was performed as described previously (65). In brief, MHCII molecules from cleared lysate were immunoprecipitated by incubation with purified anti-mouse I-A/I-E (20 pg/mg of lysate) (Biolegend) at 4°C overnight; protein G agarose bead slurry (200 mΐ) was then added (Invitrogen), with incubation for 5 hours at 4°C. After washing four times with PBS buffer, the MHII molecules were eluted by addition of 1 ml of 10% acetic acid and incubation at room temperature for 4 min with rotation. MHCII-peptide complexes were boiled at 70°C for 10 minutes. Peptides and glycopeptides were separated from MHCII by ultrafiltration through a 30 kDa-cutoff membrane filter (Sigma-Aldrich). The eluted MHCII-bound peptides and glycopeptides were subjected to LC-MS/MS analysis in order to assess peptide identity and glycan heterogeneity, as described below.

Identification of MHCII-bound glycopeptides by mass spectrometry

Reduction of samples with dithiothreitol (DTT) (in ammonium bicarbonate buffer at 50 °C for 1 hour) and alkylated by iodoacetamide (at room temperature in the dark for 1 hour) was followed by digestion with sequence-grade trypsin or chymotrypsin or by no protease treatment. After incubation with or without proteolytic digestion, the samples were further deglycosylated with PNGase F in 180 water (H2 ¹⁸ 0) in order to convert the glycan-modified asparagine to an ¹⁸ 0 aspartic acid residue. The sample was then further purified by passage through detergent- removal spin columns (Thermo Fisher Scientific). LC-MS analysis was performed on an Orbitrap Fusion Lumos Tribrid mass spectrometer equipped with an EASY nanospray source and an Ultimate3000 autosampler LC system (Thermo Fisher Scientific). Sample separation was performed on a nano-C18 column (Acclaim pepMap RSLC, 75 pm ^c 150 mm; C18, 2 pm) via an 80-minute gradient of increasing mobile phase B (80% acetonitrile, 0.1% formic acid in distilled H20) at a flow rate of -300 nl/min into the mass spectrometer. For online MS detection, full MS data were first collected at a resolution of 60,000 in Fourier transform (FT) mode, and MS/MS with CID, HCD, or EThcD activation data (all in FT mode) were obtained for each precursor ion by data-dependent scan (top-speed scan, 3 s). The resulting data were preliminarily analyzed with Proteome Discoverer 1.4 software (Thermo Fisher Scientific) and the TurboSequest algorithm. For the software search, Sequest parameters were set to allow 10 ppm of precursor-ion mass tolerance and 0.6 Da of fragment-ion tolerance with monoisotopic mass. Digested peptides with up to two missed internal cleavage sites were allowed. For undigested peptides, the minimal and maximal peptide lengths were set at 5 and 30, respectively, with unspecific search (no-enzyme group). Differential modifications of 57.02146 Da, 15.9949 Da, and 2.98826 Da were allowed for alkylated cysteine, oxidated methionines, and ¹⁸ 0-labeled aspartic acid, respectively. The search was performed against the pSyn gpl20 sequence. Any peptide identified from the preliminary software search with a false discovery rate (FDR) >1 % was filtered out. The filtered peptides were further validated manually.

Glycoproteomic analysis of recombinant glycopeptides

Glycosylation of the recombinantly expressed glycopeptides was determined by LC- MS/MS analysis. The glycopeptides expressed in 293-F cells were digested with trypsin because of the complexity of the glycosylation, whereas the glycopeptides expressed in GnTI-/- cells were profiled without tryptic digestion. An Orbitrap-Fusion Lumos Tribrid mass spectrometer equipped with an EASY nanospray source and an Ultimate 3000 autosampler LC system was used. LC separation was performed on a nano-C18 column with use of a water/acetonitrile gradient with formic acid. Full MS data were collected at a resolution of 60,000 in FT mode; MS/MS CID, HCD, or EThcD activation data (all in FT mode) were obtained for each precursor ion. The resulting data were analyzed manually with Byonic software (Protein Metrics). For preliminary data analysis, Byonic parameters were set to allow 20 ppm of precursor-ion mass tolerance and 20 ppm of fragment-ion tolerance with monoisotopic mass. The search was performed against the pSyn gpl20 sequence or the target GpepIP sequence with the human/mammalian N-glycan database (the default N-glycan database in Byonic software). Graphic representation of monosaccharide residues is consistent with the symbol nomenclature for glycans (SNFG), which has been broadly adopted by the glycomics community (66).

I-Ad binding to glycopeptides/peptides in vitro

In vitro glycopeptide/peptide binding to MHCII molecules was performed as previously described (67). Purified mouse allele I-Ad/CLIP with a 3C protease cleavage site was graciously provided by the NIH tetramer facility. CLIP was removed by treatment of the MHCII monomer with 3C protease (Pierce) for 8 hours at room temperature. The cleaved monomer with an empty binding groove was then loaded with the desired peptides or glycopeptides through an exchange reaction. Glycopeptides/peptides (50- to 300-fold excess over I-Ad) were loaded onto I-Ad in citrate buffer (pH 5.0), and the mixture was incubated for 5-6 days at room temperature. At the end of the incubation period, reactions were neutralized with 1 M sodium phosphate buffer (pH 7.5), and the mixture was spun down at maximal speed for 10 minutes to remove aggregates. Binding was assessed by running samples in pH 3-10 IEF gel (Thermo Scientific). For western blot, protein complexes were transferred to a PVDF membrane (Bio-Rad) in 0.7% acetic acid buffer for 1 hour. After blocking with 3% BS A/PBS, the membrane was incubated with purified antimouse I-A/I-E (clone M5/114.15.2) (Biolegend) at 4°C overnight. Protein bands were visualized by the addition of IRDye secondary antibodies (LI-COR Biosciences), incubation at room temperature for 1 hour, and scanning with the Odyssey CLx Imaging System (LICOR Biosciences). For MS analysis, gel was visualized by Coomassie staining (Bio-Rad). Corresponding bands from IEF gel were excised into smaller pieces, destained, and in-gel tryptic digested. The extracts were purified by passage through a Cl 8-spin column (Nest group) and profiled by LC-MS for peptide/glycopeptide identification as mentioned above. RNA-seq analysis.

Mice were immunized three times with GpepIP and pepIP at a 3 -week interval. Three weeks after the third immunization, splenic and lymph node cells were isolated and stimulated in vitro with GpepIP and pepIP respectively for three days. Antigen-responding CD4+CD69+ T cell populations and CD4+CD69- non-responding cells were sorted using the flow cytometry- based cell sorter FACSMelody (BD Biosciences). Total RNA was prepared from these cells using a Quick-RNA Microprep Kit (Zymo Research) according to the manufacturer’s protocol. RNA quality was assessed using an Agilent RNA 6000 Nano Kit with an Agilent 2100 Bio analyzer (Agilent Technologies, CA, USA). Library construction and RNA sequencing on BGISEQ-500 platform were conducted at Beijing Genomics Institute (BGI).

The raw sequencing reads were filtered before downstream analyses by removing low quality reads, adaptor-polluted reads and reads with more than 10% of unknown base. HIS AT (hierarchical indexing for spliced alignment of transcripts) (68) was used to map reads to mm9 reference genome. Gene expression levels were quantified by RSEM (69). Gene expression cluster was displayed with java Treeview 70 using cluster software (71). The DEseq2 algorithms (72) were used to detect DEGs between groups. Genes with fold change > 2 and adjusted P value < 0.05 were considered as significantly differentially expressed. With Gene ontology (GO) annotation, we classified DEGs according to official classification and performed GO functional enrichment using phyper, a function of R (73). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was also performed by pathway functional enrichment using phyper, a function of R.

Neutralization assay.

Neutralizing activity of antisera against tier 1 and tier 2 HIV-1 viruses was determined using a luciferase-based TZM-bl cell assay as described elsewhere 33. Antisera from adjuvant, GpepIP and pepIP primed and three BG505 boosts were tested against Murine leukemia virus (MLV)-pseudotyped virus, tier 1 A MN.3, tier IB JR-FL and tier 2 BG505/T332N pseudoviruses produced in 293T cells. Neutralization titers (50% inhibitory dose, ID50) were calculated as the serum dilution at which relative luminescence units (RLUs) were reduced 50% compared to virus control wells (no test sample). MLV-pseudotyped virus was used as negative control for non-HIV-specific inhibitory activity in the assay. Monoclonal antibody CHOI-31 was used as positive control (shown as antibody concentration).

BMDC induction and trimer uptake BMDCs were generated from bone marrow as described previously 24. Briefly, bone marrow was flushed out from the tibiae and femurs of 6-8-week-old female BALB/c mice (Taconic Biosciences). Macrophages were removed by incubating bone marrow cells at 37°C for two hours. The suspension cells were cultured in BMDC induction media (RPM1 1640 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 mg/ml stereptomycin, 1% (v/v) MEM non-essential amino acids, 1 mM sodium pyruvate, 2mM LGlutamine (Thermo

Fisher Scientific), 50 mM b-mercaptoethanol (Gibco), and 20ug/ml GMCSF (PeproTech)) for 8 days. Fresh BMDC induction media were supplemented on days 3 and 6.

For trimer uptake assay, BG505 was first labeled with fluorescein isothiocyanate (FITC) (Sigma- Aldrich) according to the manufacturer’s protocol. Briefly, FITC was dissolved in anhydrous dimethyl sulfoxide (DMSO) at a concentration of 1 mg/ml. BG505 was dissolved in 100 mM sodium carbonate buffer (pH 10) at a concentration of 5.4 mg/ml. A 50 molar ratio of FITC to trimer was added and allowed to react at 25°C for 3 hours in dark by agitation. Reaction was quenched by addition of 1 ul of 1 M Tris buffer (pH 7). A desalting procedure using G-25 desalting column (GE Healthcare) was performed following the manufacturer's protocol to further purify fluorophore-labeled BG505. The antibody-mediated immunogen-uptake assay was modified from our previous method (24). BMDCs were incubated with 20ug/ml FITC-labeled BG505 with or without pre-incubation with indicated antisera at 37°C for 2 hours. Cells were collected and washed with cold PBS before staining for surface CD1 lc using anti -mouse CD1 lc antibody (Biolegend, clone N418). Fluorescent signal was detected using flow cytometry'.

Statistical analysis

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HIV-infected individual has a repertoire of T cell receptors that recognize gpl20 gly copeptide epitopes (Tcarbs)

To determine whether there is a human repertoire of CD4+ T cells that specifically recognize glycopeptide epitopes on gpl20, we examined the CD4+ T cell response to gpl20 glycopeptide antigens in an HIV-infected human subject. PBMC stimulated with gpl20 glycopeptide pool (described in Example 1) showed a significantly higher production of cytokines IFN-g and TNF-a compared to stimulation with PNGase F-treated, deglycosylated peptide pool (Fig. 16). Notably, mouse CD4+ T cell epitope — GpepIP — could also stimulate human CD4+ T cell response compared to pepIP (Fig. 16).

The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and/or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.